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Lecture 05. Isotopic Masses, Isotopic Abundances, and High-Resolution Mass Spectrometry

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Lecture 05. Isotopic Masses, Isotopic Abundances, and High-Resolution Mass Spectrometry
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Lecture 05. Isotopic Masses, Abundances, and Mass Spectrometry
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This is a graduate course in organic spectroscopy, focusing on modern methods used in structure determination of organic molecules. Topics include mass spectrometry; ultraviolet, chiroptical, infrared, and nuclear magnetic resonance spectroscopy.
Thermalquelle species case chemical variety molecule electron Lecture/Conference important variants Fritz-Becker-KG tool mass spectrometer type chemical element mixture organizations Index nitrogen carbon Topic sulfur proton chemische Reaktion abundant emission water Natrium soft match function radicals coupling orders of magnitude compounds Lymphangiomyomatosis stuff
Mesomerie Lebertran Alpha-Linolenic acid molecule Potassium machine Altbier wear chromatographic chemical element organizations Peek Lava soft materials orders of magnitude coupling compounds oxygen Lymphangiomyomatosis species case Propionaldehyd islands age mass spectrometer parents Calcium ammonium nitrate mixture Acetaldehyd level weight chemical formulae nitrogen burning glass carbon proton Hydrogen Cyanide Wasserstoff Natrium Deferoxamine Computer animation magnetic periodic table quenching mixing Deuterium Destillation
milk sugars Quantitative phosphorus Lebertran case chlorine molecule Schwefeldioxid specific Ham AMPs age Methane Toast Common localization mass spectrometer oil Silicon mixture chemical formulae chemical element nitrogen fluorination Peek Wachs Lauge carbon sulfur galena source abundant end Amrinone Wasserstoff Natrium medical soft compounds silicon repeat old Brom oxygen Deuterium Lymphangiomyomatosis label
chemical element bond organizations galactose multiple Weich-PVC source striking molecule Diet food Hypobromite electron compounds mass spectrometer chemical structures Brom Bromomethane Halogene Kaugummi
chemical element Kohlenstoff-14 nitrogen species rates carbon sulfur proton crystals atoms charge sulphur end emission molecule Natrium Computer animation compounds mass spectrometer old Brom oxygen sore X-ray crystallography
Lecture/Conference Embryo multiple sulfur abundant
compounds multiple ruthenium
yeah right will good morning again so today what I'd like to talk about is 2 of 3 really important concepts in mass suspects a last time we introduce respect we talked about how the technique work we introduce 1 big idea in the big idea was that you had to have an eye on In any I'm aspect we got that ion from taking out an electron and then we talked about the CIA and other soft ionization techniques like moldy electrospray ionization and we said the big idea there is you get nigh-on by adding a proton you're adding a sodium to the molecules so the story service being concepts in respect really current very hard in fact nothing is hard about mass spect except we think differently then we've sort of gotten used to thinking as organic chemist so the 1st concept is exact manners ever since the you were well taking high school chemistry you got used to the molecular weights of compounds in calculating them based on the average mass in other words carbon has carbon 12 of the 13 CIA haven't managements of carpet but here each on being separated individual in other words you're separating each ion from each eye which means you're going isotopic from isotopic and so we need to get used to the idea of thinking about what the masses are of the individual isotopes for some of the implications of that so this 2nd big idea which ties into that is high isotopic abundance sees the fact that different elements have different different abundancies of different isotopes carbon for for example has 1 . 1 per cent of its carbons as C 13 and the rest of them as part of 12 Our sulfur has a little bit of S 34 in addition test too and will explore the implications of this and then in the last lecture will talk about fragmentation so this will be on Wednesday's lecture will discuss fragmentation as it pertains to the I'm aspect which is really a subject or a special topic in its own right we are already in the CIA in the electrospray ionization last time I saw an example of fragmentation of 80 Quasimodo actor Ryan of appropriated species remember the type of electoral and you get from soft ionization chemistry and that's essentially just Potecasi chemistry what we saw was a reaction that involved a leaving group approach needed nitrogen leaving that's chemistry that you've really been used to since your staff for a year but when we talk about fragmentation Yunnan aspect will be talking about fragmentation of radical cannot and given the fact that in soft ionization techniques you don't generally get a lot of fragmentation fragmentation isn't super importante but in the I'm aspects were you have tons of fragmentation you often can't even see your molecular it's very important foreign so I've given a couple of resources here which you'll be using in your homework assignments these are also linked to the Web page for the costs and you don't have to type in all of this stuff into the computer you can just click on the links that I provided and as you go through the homework you get familiar with using these tools and choosing choosing proper setting what I'd like to do there now and today is to really talk about the concepts behind these the 1st issues over their isotopic matches and isotopic abundance things and I thought maybe a place to to explore this since we're talking about mass spec techniques last time we talked about a variety of techniques he I'm aspect yes I'm aspect see I'm aspect is to introduce a variant of any of those techniques any of those ionization techniques that so often referred to as HR Becker higher-resolution respect In general when you get a mass spectrum the that you get is good too within a few tenths off you hundredths of a mass unit in other words if you get a number like them In 1 particular exercise show later on the homework you have a number 12 hundred 39 . 2 you might say OK that number 1239 . 2 was probably good to within a few tenths of a mass unit however with specialized instrumentation calibration you can do far better so we're very instrumentation calibration you can often get mass is to very high precision of McManus to charge ratios 2 of well I'll say high precision a typical you maybe 5 million mass units were aware of 5 parts per million were better and I'll show you what I mean by that In the case of a technique called ion cyclotron resonance mass spectrometry sometimes even 1 order of magnitude better than that so for example by 5 parts per million I mean if I had a massive 300 points you lose you lose 0 0 than the massive a small molecule maybe like a steroid or something like that we suggest that within 5 parts in millions in other words within 20 0 0 0 1 5 warm water mass spectrometry Astwood referred to as 1 . 5 m and you were merely units and in this particular case you can start to distinguish among different species that have nominally in other words to the unit mass the same Mass so the 1 thing I mentioned this before Is that your molecular weight is going to be dictated to find your isotopes president so let's take
a moment to talk about isotopes in the masses so if I go What can I look at periodic table over there and I looked at carbon and and I look under atomic weight now see that the atomic weight is 12 . 0 0 0 0 1 1 1 5 depending on how many digits from sorry . 0 1 1 1 1 5 depending on how many digits they get however carbon is a mix of carbon 12 In carbon 13 it's 98 . 9 per cent carbon 12 and 1 . 1 per cent carbon 13 the mass of carbon 12 is set by definition as 12 . 0 0 0 0 was many zeros as you wish to to to write the massive carbon 13 massive carbon 13 is 13 Kuwait all 3 sex so when you go and say I want a way out of mole of carbon or I want a way out of mold of carbon-containing compound what you're really doing is taking this number which is the weighted average of these 2 numbers in other words 98 . 9 per cent of this number and 1 . 1 per cent Of that number but as I said You're separating your molecules isotopic from isotopic America and say you're going to get a peek that corresponds to the isotopic that's all see 12 other elements also have isotopes of hydrogen for example if you want your atomic late in the periodic table is 1 . 2 0 0 7 9 4 and hydrogen is a mix of hydrogen and deuterium sometimes called heavy hydrogen it's mostly mostly mostly hydrogen 98 . 9 8 4 per cent H 1 and only . 0 1 6 per cent staged and so when you're thinking about mass spectrometry you want to use the masses of each 1 and the massive H 1 is 1 .period all right 7 8 3 further common elements that you encounter an organic compounds and nitrogen the atomic weight is 14 . 0 6 7 0 0 0 0 6 7 since 0 0 there and yet nitrogen is a mixture of in 14 10 and 15 it's 90 9 . 6 2 per cent and 14 n . 0 3 8 per cent in 15 years and so the atomic mass other in 14 of the mass that you would use in thinking about me aspect is 14 . 0 0 3 0 7 oxygen riding out the elements of this right fold again I'll give you the massive you're used to the atomic waste but this 15 . 9 9 9 4 and yet oxygen is a mixture of oxygen 16 oxygen 17 an oxygen 18 oxygen 16 predominates and 99 . 7 6 per cent there's just the tiniest smidgen of oxygen 17 . 0 4 per cent in just a little bit of 0 18 . 2 0 pacific and so the menace when you're thinking about aspect is 15 . 9 9 4 9 1 4 oxygen foreign so we've gone through some common elements here let's take a moment to explore the implications of this and see what is really valuable why the concept of exact mass is really valuable so what stake to simple molecules will take propane marriage storing CH 2 the CH 3 and will take SNL the hide CH 3 Ch so the balls a nominal molecular weight of 14 2 a 44 per day but With a high-resolution mass spectrometry you can tell these molecules apart and so let's take a look if I have seen 12 age 8 if I have seen 12 3 and H 1 8 In other words the predominant isotopic Mayor of propane then we will see that its exact Mass is equal to 3 times 12 . 0 0 0 0 was many zeros as I choose to write plus 8 times 1 . 0 0 7 8 3 when I tally that up I get 44 . 0 6 2 respects if we compare our propane to wear acetaldehyde I'd seen 12 remain isotopic was going to be 12 2 H 1 4 0 16 and the exact is equal to 2 times 12 . 0 0 0 0 except plus 4 times 1 . 0 0 7 8
3 plus 15 . 9 9 4 9 1 is equal to 44 . 0 1 2 6 2 so the point is that there is a significant difference at the hundreds .period In between the 2 different molecules that difference is 36 . 4 m you know which actually has a lot as far as high resolution mass spectrometry and what's nice is you can do more than just distinguish 2 molecules furry from each other for a small molecule you can have a computer and give you all the molecular formulas that fit the exact Mass so for example if you go to John Greaves and give him a small molecule that you've set the size and you get a high resolution mass spec on and you get a protected the exact masses he can give you all the molecular formulas that fit within 5 or 10 million mass units or you can go to that website I mentioned went on duty home page for the costs around the yard the materials for the costs and you can go ahead and say I have an exact menace of 44 . 0 2 6 2 and then you give it some parameters and you say Give me all of the compounds that fit within 5 parts per million or within 10 parts per million you don't want to give it to wider range of don't wanna give it half and half of molecular weight unit Georgia that many compounds and you say those compounds could have up to 10 carbons and up to when you pull down from the menu say 40 hydrogen up to 5 oxygen isn't up to 5 regions and you'd find out and for a molecule the small you'd find this is the only fit or you'd find maybe just a couple of its but then some of them would have absurd formulas like 5 nitrogen 1 carbon 1 hydrogen 2 right what's your questions at this point I got beautiful question while you're in the eyes so the question was if you're doing positive ions do you need to worry about adding the exactness of sodium so Of course the I'm mask spec also gives you positive ions but there you don't have a sodium a proton but in the case of electors Freddie you probably do have a sodium or proton or potassium on there so absolutely then you need to think about adding the exactness of sodium or potassium or 1 more prone to and if you have a multiple charged species say with 2 protons on there so mass to charge that's based on being a can and then you dead in 2 protons at 2 sodium sir sodium in 1 proton In a great question at all this is only if you're looking at the parent P and with alike with the I'm aspects sometimes John Greaves will come back and say this is for a fragment but it's a logical fragment of your molecule workers with soft ionization usually now typically in small molecule mass spectrometry the Journal's basically say above a thousand molecular weights don't even bother to do high-resolution mass spectrometry because for small molecules like 300 you really will get unique formulas but 4 were much bigger molecules you may have many formulas that fit now what's interesting is without cyclone resonance techniques you get about an order of magnitude higher precision so I was reviewing the paper at all of the exact masses fit To the last decimal point within a within a few tenths of a Millie mass units and my 1st thought was this guy must be cheating you hear somebody must be doing fraudulent research John Greaves and I said What could be giving such precision he said that they have an ion cyclotron resonance prospect Isobel that enlisted in there In their materials and methods section but I went to the website it's like them sure enough they do and would back technique because they're your spinning the islands around the longer you spend them the more precision you get you can get that level precision this is that's a similar idea yeah so Ireland cyclotron resonance was a detection method we didn't talk about sort ion trap can go ahead and basically be used in mass aspect mass aspect where you 1st isolate the islands of a particular menace and then you further collide them a fragment them but now I see are a detection technique what you're doing is basically having a little cyclotron a little uh magnetic field and of varying frequency in only ions of a certain frequency will circulate in residence since you 2 new residents and you basically said OK because we're right on residents were getting out of this I know this actually was in the UK last elected I have heard his name it would it's it's a pretty neat machine and in fact 1 of her professors who now retired to start his own company was actually developing ICR right here he was on the 3rd floor of this building he had a superconducting magnets 1 of my Laboratories was on the 4th floor and any time I put a computer with a CRT monitor in that laboratory the CRT monitor would get messed up by the magnetic field from his superconducting magnets In making a better mass
spectrometer and new mass spectrometry technique becomes something that 1 does for a PhD thesis 2 other thoughts a questions seems a good ones are so take a message is really simple think about the main isotopic are and let's not talk about the 2nd concept isotopic abundance and again it just takes a moment to wrap your head around this it's not nothing profound if for example I take methane and going sketch out in I'm aspect of that you'll see aligned at 16 and then you'll see a 2nd line at 17 and that line will be 1 . 1 per cent self idea of understanding give these scores of the drawing outnumber these are just give the relative intensities as 100 and 1 . 1 so we call this the and was at 1 PM and of course the M plus 1 Kate comes because you have seen 12 and see 13 in 100 201 . 1 racially you have a minuscule amount of deuterium but it's really negligible for almost anything because you have hydrogen to deuterium in 100 the 2 . 0 1 6 so essentially even if you had a molecule With 100 hydrogen saying that they would only contribute 1 . 6 per cent To the 1 but if you had a molecule with 100 cartons in it now Durant plus 1 peak would be just about as bigger maybe even a little bigger then MP so let's by comparison look at methane CH 3 C H 3 so now you see a peek at 30 and that peaks relative intensity is 100 and you see a peek at 31 and its relative intensity is 2 . 2 0 so that's your M 1 peak In your M plus 1 peak then has a probability of 1 13 we'll just forget about having heavy hydrogen because it's essentially mail but the probability of 1 C 13 in your molecule is approximately equal to it's not exactly equal to because this this is not how you do statistics but it's approximately equal to 2 times 1 . 1 per cent from 2 . 2 per cent now a minuscule number of your molecules have to see 13 and so you're implies to feet you would expect to come from the probability of to receive the teams which is kind of the 1 . 1 per cent of 1 . 1 per cent the is .period 0 1 2 per cent so another words for carbon molecule you're not going to see an end plus 2 if you want to 100 carbon molecules you'd have a significant probability of having to see 13th in other words some molecules would have no see 13th some would have won some would have to to some would even have 3 and you get nice pattern reflecting what's your comments at this now OK great question the peak that you call in class is going to be the the lowest isotopic so for example if you had a compound with 200 carbon you would see a pattern where you saw another kink another peak and I'm not giving you the exact pattern but something like this and this would still be you're amp this would be urine 1 employers to and so forth and of course if you're dealing with a soft ionization technique then your 1st speakers can be employer a Charamba sodium but basically the 1st isotopic repeat may not be the biggest in certain cases other thoughts your questions alright let's take a look at some other elements with Isetan so we've already talked about carbon about hydrogen about nitrogen about oxygen all of these are common in organic compounds let's talk about silicone solar time consists of an isotopic mixture of silicon 28 silicon 120 knowing so silicon 30 the ratio is 100 to 5 . 1 0 just 3 . 3 5 sulfur and other common element consists of a mixture of sulfur 30 sulfur 33 In sulfur 34 In a hundred 2 . 7 8 just 4 . 4 0 now what's interesting about sulfur and silicone is if you look at them they can whisper in your ear because 1st small molecule would just a few problems and that you're not going to see a significant plus 2 but if that molecule has a sulfur in it or if it has a soul economy you'll be able to pick out a little land plus 2 peak and it will whisper in your ear hate there's something up
here not all elements whispers some of them scream chlorine is 1 that screams chlorine consists of a mixture of scoring 35 and chlorine 37 in 100 232 . 5 ratio so chlorine by having a very pronounced and plus 2 screams out at you have a look at me I've got a chlorine here bromine also screams at you with that to Weissert toast bromine 79 and bromine 81 in 100 the 98 ratio you have a very substantial and was to appear other commonality elements don't say anything to to you phosphorus and I said all have single isotope there are of course other isotopes of these elements flooring has an isotope of of fluorine 18 but its radioactive and is only generated transient where you wouldn't find it a naturally occurring molecule you would find it only if you put it in as a radio label and immediately injected into a patient's safer PET imaging phosphorus has passed 1st 32 and phosphorus 33 but those also are radioactive with short hair flights and the use just for imaging and and medical and violent local purposes did Friday Dean alright so as I said sulfur in SMI gives small but noticeable and plus 2 peaks so let's take a look ,comma maker of a mole sketch here of a compound with line at 64 With all of 100 relative intensity line at 65 actually offset by drawing on this and line at 66 the line at 65 has . 0 9 the line at 66 has 5 . 0 and so the question is this is a real statue of a real EDI mass spec and so the question is His what's the company can we have Dumas so what's the Cynthia missile the thing 15 sulfur oxygen lower at 13 to 16 48 30 that doesn't work out 2 big and we'd expect and plus 1 peak with 2 carbons to be a little bit bigger anyone else Bloomberg got sulfurous silicon 1 all along sulfur dioxide and what people think that's works are equivalent to 16 6 teams that that 64 are what about the isotope pattern pretty consistent you have your sulfur giving rise to a little bit more here now I've given you the numbers based on the exact isotope patterns oxygen contributes a teeny tiny smidgen of 0 6 of 0 17 and it contributes a teeny tiny Moss manager of 0 18 right we said that there's about . 0 2 per cent 0 18 so that boosts us above the 4 . 4 per cent for the sulfur so in reality you probably unless you were doing specific quantitative mass spec for isotopes which is a whole field in itself unless you were doing that you'd probably just glance at the Spectrum and say OK I see employers to Pete something's up what fits that aspect I bet there's a sulfurous silicon the scratching your head and doing exactly what you did and coming formula the plus 1 would be from the little bit of sulfur 33 and a teeny tiny bit of 0 17 so just erase the numbers but it's sad it's . 8 cent Dad sulfur 33 and . 0 4 per cent a minuscule amount of old age 17 Our chlorine compounds give a large ship em plus 2 peak so three-to-one ratio roughly 4 1 chlorine Salemme sketch out another mass spec will start here I'll try my best to give you the relative 10 and to represent them represent them with some accuracy here Baron so 36 with 100 37 with 4 . 1 38 With
33 and 35 12 is the relative intensity yeah who cool and I write this is the game who am I HCl . now explaining are excellent start let's start with the guy here what's the big later ACL with 35 and if you remember I said that the way you generate a molecular ion will talk more about fragmentation but in the I'm aspect where you generate molecular Ryan is to kick out electron OK what about what about this peak here so that's the 13 35 that's the CL 35 feet so what about what about the peak at 38 that's the 37 1 scenario so far so far so easy OK now we've got this guy over here Cl what Seattle 35 what I plot and again to an organic chemist it's much more disturbing than to a freshman to be writing these sorts of structures will talk more more about fragmentation but with all the energy that's gone into the molecule the bonds a vibrating with electron knocked out the bonds weakened and so the molecule flies apart to give you H . plus wants and you don't Ch .period because it has no charge and then the peak at 37 38 37 plus Of course bromine compounds the very large implies to peaks so you can say by i.e. the at 2 AM plus 2 is a 1 2 1 pattern for 1 bromine let's strike let's try 1 more example here then in the I'm aspect so let's say were at 94 with relative intensity of 100 you also have 96 with relative intensity of 98 and you've got a little diet 90 5 with intensity of 1 . 1 In the little guy at 97 also with relative intensity of 1 . 1 giver tape who am I methyl bromide and of course here is you're seeing 12 br 79 isotopic here's your see 13 br 79 isotopic here at 96 is your seat 12 R 81 isotopic Merck and here at 97 ususally 13 the aura 81 isotopes so if you have multiples CLC and B are you can have in last 4 it was 6 peaks etc. for example you will get different patterns you have to Korean's in a molecule you'll see a peak and then another peek at plus 2 that's about two-thirds of the height and then a peak at 1 . 4 Source a employs 2 will call this 9 2 6 employs 4 called 1 so much of spectral analysis is about pattern recognition it's not about numerical analysis like a computer it's about looking at something in the human brain is fantastically good at picking up patters when you look at something and you say Hey this looks interesting there's this really big employers to pay that I wouldn't expect this really big plus for peak and then you think Oh it's gotta be some elements with some predominant with some substantial amount of isotopes if you have to bromine then you'll see a pattern that goes in in about a 1 2 2 2 1 ratio you're animal the employers too endure plus 4 so there's a caution here caution is that Paoli halogen 88 compounds if Eminem plus 2 were a femme plus 2 words bigger then a you might not even recognize him Marseille caution if employers too is bigger then family you might think it's demand that confused he and I'll just show you 1 more 1 more pattern 3 bro means would be a good example where you might look at this and say Oh my goodness what a wide waterway have here and so if you have 3 grown means In roughly a 1 2 3 2 3 2 1 ratio you'll see plus 2 employs 4 and 6 showing that take 1 moment too show you something from the homework just to get you to start to think about mass spec homework I homework is probably from the more on your mind right now but you're
welcome this happens to have been a problem from an old midterm exams and it wasn't the compound that was actually synthesized in my in my laboratory wasn't made to drive him 203 students crazy but rather rather for purposes of a project where we hope to grow crystals of a compound for X-ray crystallography and it's often advantageous to have brought me into the compound and so here's a here's a mass spectrum it's in ESI mass spec of a compound in which we have 168 carbon atoms 2 whirlwind Adams 38 nitrogen atoms 42 oxygen atoms and for sulfur atoms and this is the sort of pattern of
isotopic murders that you might say you know the 1st thing that I might 1 does in looking at the spectrum and say something something interesting here you look at the spectrum and you say the lines are space by unit what's going on there it's Tripoli charge the lion's share space the 3rd of a mass unit apart . 3 apart and every now and then .period for four-part so this is this is a plus the rate of charge In assuming that no sodium sore on the molecule assuming this is just charged by India's I remember we said you can pick up proton so you can pick up sodium what is that tell you about what we would call this species how many how many Proton is picking up the story so the species that we're seeing here is mn plus 3 h the blast there is talked about bromine conceded you're getting lots and lots of being peaks here bromine is going to be contributing to M plus 2 but tell me what this line over here the 2nd line is saying to the 2nd line is bigger than the first one What's that what's that telling you in this 168 carbon content there's a lot of C 30 by the time you get up to compounds that have about 100 carbon atoms in them the end plus 1 p is gonna be as big or bigger as you get beyond 100 then the empty because the odds of having a molecule with mold to put it another way most molecules are going to have it leased 1 C 13 the odds of having a molecule with 1 c 13 year 2013 is higher then the odds of a molecule having a molecule windows things whatever the elements are going to be contributing so you could have 1 C 13 you can only have 1 unusual isotope for this 1 you could have 1 C 13 what other important contributors are there going to be needed to speak at 12 39 . 5 nitrogen and sulfur and which will be a bigger which will be a bigger contributor nitrogen or sulfur "quotation mark has on that same 38 and so the .period points roughly the percentage of molecules of the fraction of molecules with 1 in 15 is going to be roughly . 3 8 per cent times 38 the percentage of molecules with oneself 33 is gonna be what so nitrogen
is actually going to be a bigger contributor to this speaks to the biggest contributor is going to be 1 13 the next biggest contributors going to be 1 in 15 and then down the line you'll have maybe 1 sulfur 3 alright well that will get you started on that last problem it's all conceptual it's always something that just involves really exploring this idea
of what exact masses and isotopic abundance is we have at hand and for those embryos yesterday and that looks more like an enema multiple and for those of you who go absolutely
fascinating patterns from ruthenium compounds and eyes compounds and compounds with multiple Ruthenians multiple lost millions