Lecture 04. Mass Spectrometry: Theory, Instrumentation, and Techniques


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Lecture 04. Mass Spectrometry: Theory, Instrumentation, and Techniques
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Lecture 04. Mass Spectrometry.
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Nowick, James
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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.
start talking about mass spectrometry and today we're going to talk a little bit about how the technique works on our next lecture on Monday we're going to talk about concepts and then on Wednesday will spend 1 lecture and EDI fragmentation which is kind of special topics it used to be really really central to mass spectrometry it's sort of part of pedagogy that's carried on by the young I'm aspect is the historical 1st and aspect commentary but is a lot less important these days mass that is a superb portend technique on molecular weight molecular formula some of the most fundamental
things that you can you can get an aspect is easily a technique to give you molecular weight we'll talk about high-res mass spectrometry from that you can get molecular formula talk about that next time in the concepts that are associated with that but the 1 thing that mass that can easily easily easily talk to you about these elements present and this is really important because you can easily see bromine and chlorine and you can see sulfur and Silicon if you know what you're looking for and what's valuable about that is Amara is not going to be a technique that talks to you about elements like that I R is not going to be a technique that talks to you so this is why you should be reading the inspector metric techniques in these days of mass that can also be incredibly valuable in getting structure it's in fact become central to biomolecular mass spectrometry to us sequencing peptides and proteins but also for more traditional organic structures and natural products you can get structure through fragmentation patterns which tries as I said we'll be talking about a little bit on our 3rd lecture you and and as I said biomolecular cases through /slash techniques through techniques like MSN mass where you're actually taking nylons and deliberately crashing into them and then smashing them and see how they break alright the basic principle of mass spectrometry is super super simple White beginning physics the basic principle you and I
love making these very simple-minded drawings of scientific instruments because it's a good way to get into our heads how the basic technique works so if you want to think about the basic technique you can think of an ionized molecules and that ionized molecules moving along until you come to some sort of magnetic fields in the simplest and historical realm is literally an electromagnetic and as the particle moves into the magnetic field its past debts then you have a writers all that right hand stuff from from physics the degree of deflections depends on demand to charge ratio no other words any given particle whether it has 20 a M U N 1 charge war 40 you on and 2 charges is going to get deflected the same amount so it's the minister charge ratio that you're seeing on the x-axis not in intercity not mass this becomes particularly important when you're doing the I'm aspect which we do a lot of fear the facility I'm sorry yes I like to standardization aspect and you do it on a reasonably big molecules were many times you get more than 1 charge on a molecule the degree of deflection depends on the mass to charge Horatio not surprisingly a heavier each of the each of the early E R I can't spell today it is deflected last the heavier particle is more massive so it's going to get that lasts more charged is going to be deflected more it's amazing how easy it is for people to lose sight of these principles particularly when you're starting to talk about fragmentation in that everything you see in the mass spectrum is going to be charged in other words of free radicals are done that has no charge on it is invisible something has to have a charge most of the mass spectrometry you're going to do will be in the positive ion mode in fact that's all we're going to talk about today but 1 can also do it in a negative ion mode where you're looking looking for negative the most of the molecules that 1 works with don't have a charge so the 1st question is how do you get a charge on the molecule historically the 1st technique developed is called electron ionization you'll see that written this year where you'll see the whole technique written as EIA mess that In the basic idea is a little counterintuitive you're going to use an electron to ionize the molecule so far so good when you have a molecule you fire and electron you accelerate electrons and give it a good hard once counterintuitive when you give a molecule good hard with an electron you knock an electron out of it so you get a Cadillac electrons where virtually nothing compared to molecules so for all intents and purposes the mass is the mass of the molecule so for example if you take the famous C H 4 and you hit it with an electron you get a CH 4 plus you've taken an electron out of it so you're getting a radical Fatah what mass spectrum call molecular ion under 2 electrons the hazard organic chemists we have trouble thinking about hot electron species most of the species we deal with have even numbers of electrons in fact I think by the time a student has taken some of organic chemistry it gets more perturbing this year's structure like this when they're freshmen because as a freshman you just learn OK you count up the number of valence electrons from carbon you count double number of valence electrons from hydrogen you take away electrons and so a freshman confront did when the problem of writing a series of Lewis structures and residents structures for a molecule like this will dutifully go ahead and say Well OK we've only got 7 electrons so I guess we've gotta make do with our 7 valence electrons that I can write a resident structural like this and I can write a resonant structure like this and I can write to more I'll just write etc and we have and that's positive charge but by the time we get to organic chemistry gets perturbing to think about this if you like to think in orbitals you can think OK we're just not being an electron out of the highest occupied molecular orbitals and you can just think of this species and say OK instead of having a field highest occupied molecular orbital we have filled out and signed by conceptually it gets easier when you have obvious orbitals when you have things that you can see rather than molecular orbitals so in the case for example of anything with a lone pair such in ether if you go ahead and you take away an electron that's minus the minus if you take away an electron from this you can say OK it doesn't look very good but there's my molecular ion there's my radical cattle and if you have an out team you can say well the pie Biddle is the highest occupied molecular orbitals so we're going to take an electron away from it I can write a resonant structure like so In the
2nd resonance structure media perhaps a more minor contributor where just what the charge and the odd electron what's your questions exactly later on when we started so the question was about not being able to see a radical so when we start to talk about fragmentation you'll see a little bit of this because at the end of today's class even showed you yes I mass spectrum molecule does break apart when 1 of these radical cannons breaks apart into 2 halves 1 will end up with an even number of electrons and a positive charge the other half will end up with an odd number of electrons and no charge and the radical because it doesn't have amassed it doesn't have any charge will be deflected and won't show up and will be detected because the detection depends upon detecting electrical current so for example later on we're going to see that if you have an ether White while make it simple like Diane Foley there you and this molecule breaks apart because when you give it away with an electron you put a lot of vibrational energy you've done double damage to the molecule you decrease the number of bonding electrons you weaken the bonds in molecule and you've put a lot of kinetic energy into the molecule in the form of the impact from the electron so the molecule now is vibrating it is hot and it has a tendency to fragment and so for example if die ethyl ether fragments and I can write curb their Americanism for the fragmentation will talk more about it later you get CH 3 plus and you get discharge species and you will observe this species when you will not observe the radicals sense for that not letting give a little more detail on the instrumentation of an actual EDI mass spectrometers to the the what I showed me before was sort of a simplified diagram and I'll still give you a simplified diagram now the 1st thing that you need to think about is all this chemistry and this is true for all of mass spectrometry occurs in the gas phase fact for common techniques an organic chemist would use the only experiment where are you doing in the gas phase II are you could do gas phase II are but most of the molecules organic chemists work with are going to be in the liquid phase of the solid phase or the solution so the 1st problem is how you get the molecule into the gas phase so typically what you do is you have a heater oil of wire like a filament and you put your sample monofilament and you have this in a vacuum and it's going to need to be a pretty good vacuum at least by the time the molecules flying along the ionization Park and have some pressure to it but by the time the molecule is actually moving you've gotta have movement in the vacuum without colliding into other molecules in fact 1 of the experiments that people sometimes do Art collision all experiments were here deliberately trying to stop the motion of the molecule but short of those experiments you need the molecule in the vacuum of you then have to see have to get into the gas phase already this means that the I'm aspect is going to be limited to molecules that can be evaporated that means that by the time you get a very big molecules like strict knowing which are going to have a very very low vapor pressure is even at high temperatures you're fighting getting it into the gas phase because of you he did a lot to get into the gas faced a general basically cotton decomposed molecule once you get the molecule into the gas phase you hit it with an electron beam the electron beam typically ends up being at 70 electron volts the molecule now in the gas phase is ionized but it's not moving at any particular rate so then what you do he is you have a pair of accelerating plates those impart velocity to of the molecule and then as I said we will in what's called a magnetic sector instrument the oldest sort of instruments have a magnetic and electromagnet the molecules will move in and depended on their mass to charge ratio will go to win detector and the detector basically measures electrical signal the molecules are charged and so you get a current and you can amplify that current and therefore said that current onto to a recording device or a computer so this is called a magnetic sector instrument and what you typically do will be very the magnetic field and in doing so as you increase the magnetic field those molecules that'd deflected blasts will then get deflected and a few plot verses magnetic field the current then you basically get a graph and that Graf translates to master charge ratio as a person's intensity so the x-axis will see and on the Y axis you'll see intensively Of the current and of course you'll see some patterns associated with the molecule and with fragments and with isotopes that will talk talk more about that in a moment and this will be called a mass spectrum and in no way technique is a misnomer because of course the mass spectrum is not a spectrum of from the earliest points your learning science in school you learn that spectrum is electromagnetic frequency and here of course this had only looks like a spectrum it looks like an animal or spectrum where Europe frequency on the x-axis in Hertz which translates the parts for millions on IR spectrum where you have frequency in wave numbers which is just a frequency unit or UV spectrum where you have frequency away what James the while very simple if you have been plus a charge going to a detector that means you've got electricity going in and so you send that to an amplifier just like microphone like a microphone in yourself up cellphone generating minuscule current and then it goes to an amplifier and then gets broadcast section gets digitized case it gets broadcast goes amplifier to a computer section a modern system would go to an analog-to-digital converter and then it goes to to a basically printer
but in the older systems of course you would go to an amplifier and then a strip chart recorder because you could literally just go ahead and have a needle go on another electrical signal to write on a piece of paper good question other questions there are lots of variations and if you talk to John Greaves he will wax John Greaves runs them aspect facility we have 1 of the premiere of mass spectrometry facilities in the country's pride best and on the West Coast here because of John's innovation in putting together a really great facility with a whole bunch of instruments and great support open access and you can go there . 4 7 if you talk to him he will wax poetic about different sorts of detectors and so forth so for example another detector that's used is called a quadruple detector the idea and the quarter detector you have for electrical rods for metal rods the ions come into the rods the detectors at the end you have alternating current of varying frequency and the rods and ions of different mass charged to charge ratios get through at different times as you frequency and so quadruple detectors is another way rather than a magnet another way that you'll see is called time of flight TO left in the basic principle here is that when you accelerate particles across a certain voltage if they're heavier they're going to be moving more slowly and so will take longer to fly and that's used in particular with with various laser techniques like matrix assisted wasted desorption 1 of the problems with the I'm aspect is that you put a lot of energy into the molecules often they fragment so often you're not seeing the molecular ion you're not directly getting a molecular weight bigger inferring that from the fragments better data developed there are a whole bunch of other ionization techniques and these are important because often you get less fragmentation so in addition to the I'm mask back electrical ionization they're a bunch of techniques that are called soft ionization techniques that are less prone to fragmentation the 1st developed is CIA or chemical ionization and the big big big differences between chemical ionization and electrical ionization the big difference between all of the soft techniques and electrical ionization is instead of knocking an electron out of the molecule you're putting something charged onto the molecule often what you're doing is adding a proton to the molecule which in a way is much more intuitive to inorganic chemist because you're basically using a strong acid are using an acid of various forms the department make a molecule 1 of the problems of chemical ionization 1 of the problems of electrical ionization is over here getting the molecule into the gas phase as organic chemists and biomolecular chemists have become interested in bigger and bigger molecules the targets of organic synthesis have gotten bigger people are interested in proteins and nucleic acids in the wake of Sakharov's as all of the molecules have gone bigger got bigger the issue of ionization has become more important another technique that's been developed is called fast atom bombardment and I'll show you more about these injustices 2nd there is a variant of this technique referred to as LSI life 1 case you doing the business that also you with an atom in the other case with an eye on In moldy and is another technique for ionization getting molecules into the gas phase it's matrix assisted laser desorption ionization I'll talk more about all of these techniques in 2nd Our so just the hind chemical ionization is dead a reagent gas is going to be used as a probe the molecule you're going to put a proton onto the molecule sometimes sometimes it'll be another ion but you'll do so with the reagent guess what you're doing is is 1st of ionizing the reagent gas but unlike the conditions were doing ionization in aspect which Avery very powerful vacuum for a high vacuum we're doing that under a week vacuum at about . 5 millimeters a mercury know what's happening under those pressures is that you're Europe Ireland's that you're generating all the reagent gas of methane ammonia ice of butane are colliding with each other and what they're doing is making assets for example as I said nothing I said butane the among the so let's look at the chemistry of the reagent gas so we saw that if you take methane and you give it a good hard whack with an electron you'd get a methane radical can you get CH he was not in the gas phase when C H 4 plus not collides with another methane molecule what happens is you transfer a hydrogen and so you get C H 5 plot protonated methane in other words you basically go along the proton onto the methane structure as you might imagine this and not all the 5 hydrogen sort of stuck happily around 1 carbon 1 sort of Guandong onto the side of the molecule as you might
imagine this is a very strong acid from the imbalance our equation we also get a methyl radicals so now when you have this very strong acids C H 5 plus and it collides with your molecule which is come off of the heater Quayle nail it transfers a proton and to the molecule to give you an age classes once each form and that's very easy to conceptualize if the molecule has a lone pair of electrons you probe made the lone pair of electrons if you haven't Easter you probe these there if you have an alcohol you program LH group to give your product made alcohol if all you have in the molecule is now keen you probably made the Albertine to give you a Koble Qatar and so we call this species acquires the molecular ion and of course the big distinction is this is in life plus 1 in other words it's 1 higher than the molecular weight and sometimes you'll see other things glomming onto the molecule including including how the fragments in see I'm aspect so methane-a gives rise to this C H 5 classes your reagent acid I said butane gives rise to a church funeral Carbo Cadillac In at 1st you might say Well wait a 2nd that doesn't look like an acid but of course if you think about it turbulent Parbo Qatar and eating give up a proton off of the adjacent carbon and give you I severely so the church group Pavel Caroline is also interested in the gas phase it's less of a strong acid than C H 5 plus this is really unhappy this is only somewhat unhappy so the ionization conditions generate a lower heat of reaction that's important because that means we're that Proton remember this is in the gas phase so when the reaction occurs and the reaction is exit the molecule is hot it's vibrating very strongly and it is still prone to fragmentation so the less energetic the ionization the less and felt that the ionization process the less energy the West strongly acid the less from the molecule is to fragmentation and the more likely you are to actually see acquires the molecular and not some fragments ammonia although we don't usually think of the ammonium ion as being strongly acidic in the gas phase the ammonium ion is a strong acid because it gets its stability in water from being solvated and here you have no salvation you don't have hydrogen bonding in the gas phase so even the ammonium ion is a strong acid in you get onerous do you get polymers station of radicals in mass because mass Petron the trees is under conducted under conditions where your molecules are not typically colliding you will not see polymer In yes I'm aspect which will talk about the moment because the molecules are actually starting in solution phase you may a pair of molecules that are already stuck together so you may for example a molecular ion that's derived from 2 molecules and let's say 3 charges but yeah you do not typically seek polarization so so you're not going to see the ch 5 plot but when you get your molecule so let's say you're molecule is dying home Easter so now what you'll see is not something that I fully theories 29 plus 29 plus 16 but what you will see that it is not something at once 29 was 29 was 16 not something at 64 from doing the math correctly in my head the way not something it 74 but rather something at 70 5 for the protonated ESA Over you see the plus 1 so I said butane acts as an acid as well and I'll draw barrel mechanism so all just Roberts is based the base take softer proton and this is exactly the microscopic reverse of the reaction that you get when you program a canal thing so you end up With severely and the age class and if you think about it if you probably made Isabella with a strong acid like sulfuric acid for example and Friedel crafts reaction the 1st thing you do is you put a proton here you get repeal probably have so acting so this is just the microscopic reverse that process good question because you have an ionization chamber 1st so you basically have a chamber where Member I showed you the electron beam so you have a chamber with methane that's at a higher pressure that set about . 5 millimeters an electron beam going into that the methane is getting ionized it's colliding and then it's diffusing into a region where you have the Iroquois all the heater Quayle and sample now the problem with see I'm aspect is you still have to get your molecule into the gas and so for a very very big molecule this may not be feasible by heating it even in a strong vacuum because the molecule may not vaporize just decompose right if you go ahead and you heat up sugar lot you don't have the sugar boiled you have the sugar carbonized and summarily for other rabbinic molecules they may simply carbonized and then you get those roasting toasty caramel smells but not the the smell of actual sugar soft ionization techniques in which the ionization process gets the molecule into the gas phase solve this fast atom bombardment was I think the first one developed and in that case what happens is you take an adamant that's moving quickly and you actually do that by an electrical processed ionize accelerate and reprogrammed a bad actor really neutralize that Adam you have your sample on a target and you have your sample In a matrix matrix is just another way of saying a viscous solvent the matrix is like cholesterol were nature been so alcohol what happens when the Adam fires into the molecule in the matrix use butter off molecules that some of which are approximated the basically have the molecule essentially get
protonated and you'll see H class for inmates major once dot matrix but the word sometimes you'll see about molecule of blistering of molecules like prevents allow alcohol complex with your molecule so this is good for our highly polar compounds in non-volatile compounds in higher molecular weight it's also good for compounds that tend fragmented CIA if you want to see the molecular ions as I said there is a variant of fans called LSI mass back in liquid secondary ionization mass spec rather than firing and Adam you're firing Ireland such as cesium plus but it's the same basic principles you put a good hard wax in there and you end up ionizing the molecule and getting it in the gas phase I don't want to give hard numbers let's say up to about 20 thousand molecular weights said this really opened up a whole new home in the realm of mass spectrometry including biomolecular master professor at all both at the techniques but fast atom was the 1 that 1st was popularized John Greaves does the Ellis technique and again I'm sure he will wax poetic on the differences between the 2 techniques but but for your purposes the pretty similar it does I you end up having to have what you can go with stronger magnetic fields or I think typically this is done with a quadruple and then from all the which I'll tell you about the 2nd often people do time of flight because time of flight tolerates even bigger strange year on is another technique that's widely used now these are open-access instruments aspect has become very populist very cheap very easy to do and 1 of the reasons for this is because a regular EI mass spectrometers a relatively fast instrument although there are often made into parts of Gazprom paragraphs and so forth but they often require a lot of character yes I now is a lot easier to care for its goes up to to very high molecular weight and analysts say maybe 5 million but basically just very very large so the basic gist is your spraying off of an electrically charged nozzle you're spraying charge micro droplets sprayed into a vacuum and what happens is the droplets in the vacuum the insolvent like methanol the solvent evaporates the charges which are put on electrically get closer and closer together as the solvent evaporates until they repel each other and the droplet chatter apart and then you have more evaporation and more shattering and eventually you get charged species free of solvent so you often end up with multiple we charge species provide for big molecules but also a big problem biomolecules so for example you will end up with the H in class so for example you might end up with 3 protons on your molecules often you will pick up sodium so you will end up for example with a certain number of protons and a certain number of sodium Sonya molecule the sodium cat and will give charge do it as well so anyway I'm gonna show you an example of this just as just a 2nd I'll show you an example of an yes I'm aspect let me just mention molding another technique that John has in his facility so you're using a laser blasts the molecules in a matrix the matrix is a species with a coma for that absorbs the laser light and again you get protonated molecules so again you get for example in each class and again this is good for very high molecular weight by up to approximately 300 thousand molecular weight but again for a very large mn aspect has gotten coupled widely with other techniques including separation techniques so you will see mass spec on detector of a gas chromatograph you'll see mass back on the back end of a detector of liquid chromatograph for example age PLC and even see hyphenated techniques were you have aspect of coupled to mass spec were due Friday your ions in a controlled fashion to learn about their structures so as I said in the soft ionization techniques what you're doing is taking the molecule and putting a proton on it for sodium ions itself for example to give you a major cluster for example so I'll just give you too trivial examples you wouldn't typically look at methanol but it's a nice simple way to think about it if you put a proton on methanol as I indicated before when I talk about it I told believe you there you will end up with protonated methanol you have sodium from glass everywhere and so if you put a sodium on new soft ionization for example in years Simon aspect you'll end up with a sodium on your molecules and what I want to do now sometimes you'll even see so this would be an plus 1 this would be employs 23 sometimes you'll see potassium as employs 20 in plus 39 so what I wanted to show you an actual actually ESI mass spectrum of the molecule they have a number of In doubts your hand out here and I think we may need to ship to show a few handouts over extras try not to try not to chop down too many trees here but I always liked to give a few extras Our said this and we're going to be talking more about actual mass spectra and subsequent ties everyone have a handout alright said this is handout of a particular molecule this 1 happens to be an example of a
peptide now we're going to talk more less next time but 1 of the big concepts is your separating molecule by molecule which means you're looking at individual isotopic murders put simple late 99 per cent of your carbons are carbon 12 1 per cent of your carbons are carbon 13 so when you calculate amassed firm aspect you're going to
actually calculate the exact manners that's based on the predominant isotopic murders the
exact massive this molecule 744
. 5 and so
if you look at the mass spectrum the 1st speak you see here Is this peak at 7 67 . 6 here you see a peek at 7 14 5 . 7 so this speech is you're and plus H plus the instrument is only good to plus or minus a few tenths unless you're running in a high resolution mode so in other words we would expect if we pick up a proton here we get to 745 . 5 4 at 745 . 7 that's within the limits of experimental error this peak over here corresponds to implies in a sometimes you will hear the figures speak in the spectrum referred to as the basis peak in the spectrum this speech here corresponds to a 13 isotopic will talk more about that later that's a molecule with 1 the 13 in here you'll see the same over here you'll notice you even see molecules with we typically don't get a lot of fragmentation in the mass spec but you'll see for example here you have a fragment you're doing acid chemistry on the molecule what's happening in generating the fragment is your coordinating on this nitrogen and then it's leaving for that particular fragment to generate and psyllium ion and here's where the concept of charge comes in the fragment is uncharged so this is basically I'll just write etc. for the rest of the molecule were
fragmented right it is failing to quickly respond to the fragment the charged peak the charge species gives rise to this clear here and this is another fragment over here as far away as a concept that I wanted to 1 bring to mind is question the last concept I wanna bring to mind is something very simple it's what's often called the nitrogen rule and I'll just play with this for 1 2nd nitrogen role is that compounds with an odd number of nitrogen give implies In the Iron Man aspect and you can convince yourself of this in other words if you look at try methyl meaning that contains 1 nitrogen its molecular weight is 15 million if you look at try fed only if you look at the base of butane which contains no nite positions it has a molecular weight of 58 and you'd say OK that's in the I'm aspect and everything turns on its head in the soft ionization reversed so for example plus H last trifle try methyl Amin now would be 60 and if you were somehow program aiding this what you might do in the city I'm aspect it would be 60 about 59 and so again on inspection of the mass back if you look at the mass back you can go ahead and say OK this compound has an odd number of nitrogen or this compound has an even number life which is the only caveat is with fragmentation In the EIA everything can get messed stuff so for example if you take on if you take turns Putin all during the year all has a molecular weight of 74 but you're often not going to see the turbulent and all you'll often see a cargo Qatar and I'm aspect of often see interview cobbled and that has and that's a minus 17 that's 57 and so if you just looked at the peak of the biggest Pakenham mass in the Iron Mask spectrum of Turkey all you'd say all the highest molecular weight peak is 57 this has 7 nitrogen reality notes from anywhere that's something something to keep in mind is away with small molecules of saying OK what elements of will pick up next I'm talking about all other elements present we're going to talk about Korean's bro means we're going to review the concept of exact Marcel a bit more


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