 Hello, welcome back. I'm Lanak Martens from Gent University and VIP and we're at our third lecture in mass spectrometry basics and today we'll talk about mass analysis. So what we've done before in the first lecture is we covered amino acids and proteins and how the properties of the amino acids make proteins such useful molecules and how we can use these properties in mass spectrometry. Then we talked about the overall concepts of mass spectrometry and then the ion sources, which were the topic of the second lecture. Now remember the ion source was really important. The ion source allowed us to take our analyte molecules from samples and turn them into ions in the gas phase. And this was really crucially important because you need ions to analyze them in the mass analyzer. And that's where we are today. We are at the mass analyzers, our very sophisticated pair of scales that will find out how much a particular molecule has as mass. Now in order to do so, I've already told you that they use electrical fields and so we'll be seeing this throughout the presentation, how these electrical fields are being used in different ways to measure mass. Okay, let's start with our first type of mass analyzer, the time of flight. Now it's a very descriptive name again because it's going to be all about timing the time of flight of a particular ion. So how does it work? We have the source over here and remember the source is always this dark blue and the source can be electro spray or Maldi. It doesn't really matter for the time of flight. It can be either one of these. So it generates ions in the gas phase. Now these ions will then encounter what is known as an extraction plate, which is this thing here. And you can visually imagine it as a kind of chicken wire, loosely knitted piece of metal where there's a lot of holes and a few wires. Now this extraction plate is at a high voltage, it's at 30,000 volts or so. And obviously our ions are going to see this plate and the electrical field that it generates and they will migrate towards this electrical field. So in a way this is an acceleration device that given a certain voltage is going to generate a certain amount of acceleration and therefore velocity in the ions. Now what comes then is this thing is mostly hollow, remember. It's mostly open space with a few small wires in between. And so the ions are going to fly through generally and then they will enter this green contraption, which is essentially a very finely machined long tube. This tube is at high vacuum as everything else is since the source. So it's a very high vacuum tube and most importantly there is no electrical field in this tube and that's why they call this a field free tube. So it's essentially just that, it's a vacuum tube machine to very precise dimensions. Obviously the ions that have been accelerated through this electrical field generated by our extraction plate, they will have a certain velocity. And now with that velocity, because there is no resistance from the air, it's vacuum and because there is no electrical field, they will just continue with this speed and travel along the length of this tube. And at the very end we will have a detector and we'll talk about how these things work in the next lecture, but there will be a detector and that will be able to find out when a molecule has traversed this field free tube. Now these tubes are pretty long, they're typically longer than a meter and there's a reason why they're long, but we'll get back to that. So now let's have a look at a small animation to see what happens in this type of flight. Analyze. So we have our sample ions and the sample ions are being moved by the extraction plate and they receive a kinetic energy that is equivalent to the charge of the individual molecule here, or ion, and the voltage that we have applied, so the strength of the field and the charge. Obviously if you have twice the charge, you will feel the fields twice as strongly. Two plus ions will feel the same field with twice the strength of a one plus ion. Now that is the kinetic energy that these ions receive and so they will start moving with a certain speed that as you know relates to the kinetic energy. So this is secondary school education. The kinetic energy of a moving object is the mass times the velocity squared divided by two. Now we know where this kinetic energy comes from, it comes from the voltage and this voltage is something that is under our control. That means that we can figure out somehow something about this speed and this mass. In fact we can say that the speed is equivalent, or will be equivalent, to the square root of two times the kinetic energy divided by mass. It's simply solving this equation for velocity. As you can see a large ion should move slower than a small ion because a small ion has a smaller mass and since the speed will be inversely proportional to mass, small ions will go faster. Do note that there is an exception. And the exception is if the very large ion with a large mass actually has a high charge, it could actually go faster because the kinetic energy would be higher. But by and large for the same charges state the more massive ion will go slower. So speed gives us something about the mass. In fact we see a relationship between the speed and the mass. Now the way that we work this out is we work with the laws of kinetics and a little bit of calculation here. We say that the speed can be expressed as the distance covered in a given amount of time. Since we know the distance, the length of this tube, we can calculate how long it takes for the ion to traverse this tube and then we know what speed that ion had. So the speed comes from the time of flight of a fixed distance. So we know when we've switched on the extraction plate and when we switch it off. So we have an idea of when this ion enters the tube and then we know when it hits the detector. So we actually can with a very accurate stopwatch determine how long it takes for the ion to fly through the tube hence time of flight. Now that gives us velocity and as you can see velocity relates to mass but we still have the kinetic energy. So the kinetic energy is of course related to the charge and the voltage so we can fill that out here. So two times the charge and the voltage divided by the mass square root gives us the velocity and velocity of course is distance traveled divided by time taken. And when you fill all of that out in these equations which is an exercise for the viewer you can do this yourself but you should end up with this equation. Mass over charge equals two times the voltage divided by the velocity squared and of course we solve velocity by distance traveled divided by time that's squared of course and we end up with this final equation that the mass over charge equals two times the voltage applied times the time traveled squared divided by the distance traveled squared. Let's see what we know about this. We do not know mass and we do not know charge so we're actually trying to find these out. We do know two because that's rather simple. We know V the voltage because that's something that we told the instrument to apply on this extraction plate. So we have a voltage source that's regulated, very tightly regulated and we can set it very precisely. We also know the time traveled because that's our stopwatch that's built into the mass spectrometer and we know the distance traveled because we know how long this tube was to begin with and so in total we can resolve any term here but we cannot resolve these two and this is an important thing. What we see here is that while we resolve the mass over charge we cannot distinguish between different charge states of different ions so we always measure mass over charge together never mass alone or charge alone and this is a really big limitation. Now if you were to think of this, if you think of this we actually talked about this in the ion sources where the Maldi source if you will recall has only singly charged ions when you analyze peptides so there it's quite easy because Q is always one and so this whole equation becomes very simple and we are actually capable of reading the mass. For an electrospray source which are more popular because they can be ran 24-7 and automated we don't have this luxury, charge can be 2 plus 3 plus 4 plus 5 plus and we simply don't know. So this is a downside of using electrospray we have multiple charged ions and we have no way of resolving that charge from the mass spectrometry measurement. Mass spectrometry always measures mass over charge. Now mind you, I've written Q here for the charge which is the way physicists would write charge. Mass spectrometrists however don't. They use the letter Z or Z and so it's always M over Z, mass over charge is M over Z they never write M over Q. So I apologize for using the correct units here or the correct variables in physics in fact it's M over Z that is being referred to by most mass spectrometrists. Now there's another thing we should note what we are measuring here is mass and it's not gravitational mass what we are measuring is inertial mass so the resistance of a particular molecule to acceleration. Having said that to the best of our current measurements gravitational mass and inertial mass are equivalent so there might still be some differences but we haven't been able to measure the difference but it's an interesting point we measure inertial mass not gravitational mass. So that's how a time of flight analyzer works it's a very sophisticated scale that uses electrical fields and then the time of flight through a tube. There's one other caveat though and that is that this tube tends to be made out of metal and you all know what happens with metal when the temperature changes. If the temperature shrinks or goes down sorry the metal tube will shrink in size. If the temperature goes up it will expand and so this tube despite the fact of it being very very precisely machined will not be exactly the same length at any given time. Small fluctuations in temperature will create fluctuations in length and the fluctuations are sufficient to be detectable by our very advanced chronometer built into this muscle parameter. So we somehow need to figure out what the actual length is of this tube every time we use it and for that we do calibration and calibration is incredibly important if you want precise measurements. The way it works is very simple rather than resolving for mass over charge we're now going to resolve this equation for X and the way to solve for X is to put in a molecule of which we know the mass over charge so you take a known analyte where you know which mass over charge it will take in your instrument and then you use that in the instrument and you calculate back what the X should be and that gives you the actual length of the tube at that point in time. So the best way to do it is to do it with the sample. So when you take an analysis of a spectrum there should be what they call internal calibrant present in that spectrum too and then the calibrant allows you to calibrate that spectrum perfectly and therefore get a more accurate mass of the analyte. That is not always possible but people try very hard to get their calibrants as closely spaced in time to the actual analyte as possible. So calibration is really critical. You always need to calibrate your mass spectrometers and this is the reason why the other mass spectrometers, the other analyters that we will be talking about all need to be calibrated to show you here why calibration is so important. Okay. So without further review let's go on with the next analyzer which happens to be one of my favorite analyzes and it's the iron trap. So an iron trap analyzer does exactly that. It is a trap for irons. How does it work? We have the source over here and then I have this beautiful three-dimensional drawing here with a box. So imagine this is actually a box. There is a round opening on one side of the box and there is a round opening on the other side of the box. So essentially there is a tunnel through the box two openings on opposite sides. And in the middle of the box as you can see here rendered in beautiful infinity we have a ring, a central ring electrode. So take it like this. You've got a hole in the box here. You've got a hole in the box there and in the middle of the box you have this ring and this ring electrode and these two holes they have a current on them. Now the currents are a little bit special and we'll have a look at that in a second. Our analyte comes from the source or irons, you can see small and big ones and they get into the trap. Now why does the trap capture and trap irons? Because it uses a sophisticated set of voltages. It has a direct current voltage and an alternating current voltage. And the alternating one has a radio frequency so essentially it's oscillating really quickly between plus and minus. Just like your alternating current from a wall plug would do it switches sides all the time. This is exactly what happens here and it happens really fast in a very sophisticated way and what happens is the irons move into this trap and they see say a negative charge on this side and they say oh let's run to the capping electrode on that side and they start moving towards that capping electrode but before they can reach the capping electrode the field switches around. So now this capping electrode becomes positive and the one behind me becomes negative. So if I'm a positively charged ion I'm going to turn around and now I'm going to run that way. So I'm slowed down, turned around and I'm going back and it switches again on me so now I have to turn back and go back and so I am now effectively trapped. I am constantly moving back and forth but I never make it to the capping electrodes on time. Now in fact this movement happens in one dimension so it's kind of a blob of ions that are all in this trap and cannot get out. This is the basic premise of the ion trap. Now this is really nice because we can actually accumulate a lot of signal, a lot of ions so we have a total ion count that is quite high inside this trap but we still need to figure out what kind of mass they have. Now the way to do that is to again play with this voltage. We're actually going to make use of the fact that just like in a time of flight if you put an electrical field even though it's oscillating every time these ions are going to be accelerated towards one of the capping electrodes. If you're a very massive ion you're going to be accelerated at a very leisurely pace you're going to go slowly towards the capping electrodes but if you're very tiny you're going to go very fast. So these small ions are very fast and we have to switch the field quickly enough to make sure that they don't escape. Now if we would switch just a smidgeon slower then these very tiny ions actually make it to the capping electrode and out before we switch the field. So now the capping electrode is negative I'm a very fast ion, I move out I'm now out of the capping electrode which is here now they switch the field and I no longer care because I managed to escape the trap before they switch the field and only the small fast ions can do that. So if you do this consistently and you slowly decrease the period what will actually increase the period so it takes a longer time to switch what will happen is the first ions to escape are the very small ones and then they're all gone and then you make the switch a little bit slower and then the next smallest ions make it out and the next ones and the next ones so you're starting on the small side of the ions and you're moving them out until you reach the very large and slow ones so let's see what happens the small ones go out first and then the bigger ones until you hit the most massive ions and they all hit the detector so the timing of when they hit the detector related to the voltage you've set at that time it gives you an idea of what the mass would be of that particular ion now mind you this is not as accurate as the mass determination that we had in a time of flight our stopwatches are simply better and our tubes are long enough for what we call a better resolution and ion trap does not have a very good resolution unfortunately because this voltage setting is not super precise as a result this measures mass with a little bit more of an error and we'll come back to that later but it's very very useful because it traps ions and once ions are trapped you can accumulate them wait for enough ions to be there to get a strong signal so that many ions or as many as possible hit the detector simultaneously we'll come back to that in the next lecture by the way interestingly Wolfgang Paul and Hans Georg Diemelt got a Nobel Prize in 1989 for the invention of the ion trap so the ion trap has been around for longer than say, electrospray since I'm on the topic of the source again usually ion traps are paired with electrospray sources it's a good combination because the source continues to spray sample in and the ion trap can accumulate a certain amount of that and then analyze it in small sections so this is usually a combination of an electrospray source and an ion trap analyzer it's a really nice way to work with your ions now the ion trap has a cousin if you like and this cousin is the quadrupole the principle is extremely similar but instead of a cage, a box in which you trap the ions we're now going to create what you could call an ion bouncer so the principle is very similar you have four electrodes that are located in space so there's two parallel ones and then there's two above so you can actually have six or eight these days they have many more but four will illustrate and you put as we said before I now use a physics notation for this a direct current field and an alternating current field with a radio frequency and again we're constantly shifting on these rods that are spaced in 3D space we're constantly switching the voltage so I'm an ion, I enter this hallway if you like of rods and so now I'm attracted to the left rod and now it switches, now I'm attracted to the right one to the left one to the right one so I'm going to go back and forth now in an ion trap the idea was to keep ions stable to never let them escape here we kind of do the opposite what we're aiming for is what is known as constructive interference it's the reason why soldiers who are marching in lockstep that they have to break their step when they hit a bridge because as soon as you start walking on a bridge and everybody puts down a foot at the same time if it happens in the right rhythm every time the bridge is going to oscillate a bit because of the feet coming down if everybody presses their foot onto the pavement when the bridge is at its lowest point you're going to every single time push it deeper and deeper and deeper and so your oscillation becomes worse and worse and worse until it breaks this constructive interference is exactly what we want here I am the ion, I'm moving, I go towards the left then I move towards the right but I don't go as far to the right as I went to the left if I keep doing this for a few times at some point I'm going to either fly out of these this hallway defined by the rods or I'm actually going to bump into one of the rods which is actually more likely only a very specific ion with just the right M over Z mass over charge will be able to maintain what is known as an overall stable trajectory only a very specific mass over charge will be able to move as far to the left as it moves to the right and as a result on average it goes straight and makes it out of this corridor defined by the rods everything else is literally being thrown out or in this particular case thrown against the rods as I mentioned which is actually quite interesting because as you know amino acids contain a lot of carbon and when these rods are being hit by the amino acids the amino acids essentially turn to ash and so what you get is you get carbon scorching on these rods and if you have a quadrupole instrument you will know that you have to clean these rods or have them cleaned at regular intervals they actually accumulate almost like a barbecue they accumulate a layer of encrusted carbon that ultimately limits their effectiveness so these things need to be cleaned as a result as it is it's very different from an iron trap in its actual workings because the iron trap remember it accumulates all the ions and then you can analyze them at leisure here however we throw most of the ions away because only one set of mass over charges can have a stable trajectory and everything else is just wasted so this is not usually used to analyze say a full spectrum of all possible masses you could in principle do that you could say let's start with a stable trajectory for the smallest mass the second smallest, third smallest and then scan the whole mass range but meanwhile you would be throwing so much sample away the iron trap is much better for that because all the ions we're not analyzing they just stay put in the trap and we have plenty of time to analyze them because they're not going anywhere here we waste them so a quadrupole is not usually used to really scan a whole mass range or to produce full mass spectra instead it's a really good bouncer if we only care about our blue ion here we can get rid of everything else very effectively so that's why I refer to this as a bouncer like at the door of a bar you want to get in if they don't want you to get in the bouncer will make sure you don't get in so that's where these things are very very useful but they have surface mass analyzes as well and they're closely related to the iron trap in principle with the alternating electrical currents and the stable or unstable fields now we've talked about the resolution of mass analyzes and there's a very specific thing about resolution that is important when it comes to determining the charge of a particular ion remember that we always measure mass over charge and that we have this problem that the mass over charge is a unit that we measure and we cannot get rid of this charge component and in electrospray this is a problem because we don't know whether the molecule or the ion is 2 plus or 3 plus or 4 plus so it's very difficult to know exactly what's there is it a 2000 Dalton thing which is mass with a charge 2 which makes it look like as if it's a 1000 Dalton because we measure mass divided by charge or is it actually a 2000 Dalton ion that has a charge of 1 which gives us an M over set of 2000 it's very difficult to figure this out and so the way that you could potentially figure this out is if you have sufficient resolution now resolution is defined in a very vague way but you could essentially see it as the ability to make a separation between two adjacent peaks so how closely spaced can they be that we can still see them as separate peaks and this is usually expressed as a large number at a given mass but it can depend on the vendors in general higher resolution is better you may remember from the second lecture that we mentioned the digitizer and how the digitizer samples the analog signal over time and if we sample more closely we actually get better resolution and this was an upgrade to an instrument well imagine here if we sample here here and here we would never know about this graph in the data however if we would have a high resolution sampler that samples all the way through we could get a very accurate representation of this data so that's where resolution comes in I'll show you some real life data this is for a very old type instrument or an iron trap so an old top time of flight or an iron trap would look like this but we can't see the differences between the different isotopes it essentially becomes one big pile of signal and the best we can do is calculate an average mass so we try to find the middle point of this distribution and we say that's roughly where the MOVZ is do note that I use things like mass here but you all know this is incorrect right it should be mass over charge it's just a short hand and you will see a lot of people refer to mass actually not measuring mass they are measuring mass over charge so I apologize for not being very consistent but at least the literature is not consistent about this either now that's not a very good situation because in reality it should look like this these are different isotopes of a given peptide and they are very nicely separated and they are separated because we have higher resolution we have a much more detailed view that's actually there and here we can use something called a monoisotopic mass and this monoisotopic mass is the lightest isotope and refer to as monoisotopic that is a definition that comes from small molecules where this problem is relatively straightforward small molecule tends to have only carbon-12 and so carbon-12 give you the lightest isotope typically and that's where you define it I mentioned carbon-12 so you probably are familiar with isotopes and there are many isotopes in nature very famous one is carbon-14 carbon-14 is being used for radioactive dating of archaeological finds but carbon-14 is radioactive so it's not stable it actually disappears over time there are however stable isotopes that are pretty common so carbon-12 is the base isotope of carbon and it's very stable but it has a heavier brother which is carbon-13 which is also very stable and not radioactive as a result all of us all living things and even all inanimate things contain a certain amount of carbon-13 and that amount you can find on a Mandelaev table they will actually list the relative prevalence of the different isotopes and you will see that carbon-13 adds one Dalton to the mass if you have one carbon-13 of course it's one Dalton if you have two carbon-13 it becomes two Daltons if you have three it becomes three now I make this fuss about carbon-13 but you may know other isotopes you may know for instance Oxygen-18 Oxygen normal isotope is 16 that's the monoisotopic mass but there's also an Oxygen-18 which is also stable, non-radioactive so that occurs in nature and of course people know Deuterium which is the heavy form of Hydrogen Hydrogen has one mass of one and Deuterium has a mass of two so it's the proton and the neutral so why am I not talking about these isotopes here? that is because their relative prevalence is so much lower than carbon-13 carbon-13 is by far the most relevant isotope in living things Deuterium is really so low in abundance that while there could be Deuterium here is going to be negligible in the actual effects that it has on this isotopic envelope so carbon-13 is the only one that really matters now there's an interesting thing that happens here and you may have noticed this this is the carbon-12 isotope and carbon-12 is like 99% of all carbon atoms and yet this peak is smaller than that peak which is the carbon-13 you can literally read this as saying there is more carbon-13 than there is carbon-12 in this particular molecule that is actually a bad way of saying it let me try and rephrase that and say it more accurately it is more likely that's the higher intensity to have at least one carbon-13 in a molecule of this particular nature than it is to have only carbon-12 now that is actually not that hard to understand because it's a matter of simple statistics if you have a relatively big peptide and it contains say 100 carbon atoms and one in 100 is likely to be carbon-13 then it's very likely that you will find one carbon-13 at least because you have 100 carbon atoms and one in 100 in nature is carbon-13 so you will likely to have one if you have 80 carbon atoms well the likelihood is still going to be pretty big that at least one of them is going to be carbon-13 if however you have only 50 carbon atoms well then the likelihood goes down dramatically and what you see here is exactly that the bigger a peptide is that is the more carbon atoms it contains the more likely it is that it has a strong second and third isotope and in fact you can roughly do it by mass and around about 1800 Daltons so 1800 Daltons you will see an equivalence between the first isotope and the second isotope at about 2000 Daltons you will see that the second isotope is clearly bigger than the first isotope and if you go to about 2200 Daltons it will look like this go beyond 3000 Daltons and it becomes very hard to spot this monoisotopic peak and that is a problem because the monoisotopic peak is the one that we define for the mass of a analyte if we have sufficient resolution so here obviously we cannot use that so here there is no problem we use the average mass here however since we have the monoisotopic mass if this carbon-12 peak becomes so small because there are so many carbon atoms in our peptide that at least one of them must be carbon-13 then we can no longer find this peak and the peak picking algorithms will pick the next peak and call that the carbon-12 peak the monoisotopic peak but they are off by one Dalton which is a much bigger margin of error than you typically get from the measurement so you will see that a lot of software that tries to identify peaks allows for a so-called carbon-13 switch and the carbon-13 switch has a little bit of logic in it if the peptide is pretty big then imagine that this is the wrong peak that the mass you get is actually one Dalton one neutron too heavy and instead use this other mass it does have an effect here because if we do eliminate the carbon-12 peak it actually shifts the distribution a bit higher but because we take an average mass that usually means that the shift is smaller than here where it really is a full Dalton this whole long story about isotopes just to show you one other thing and now we get back to the charges I promised to talk about is that since we know that the difference between this peak and this peak should be one neutron and the distance between the second peak and the third peak is again one neutron we can measure how much distance we actually see because the M, the mass is one Dalton or one neutron but if we measure half and mass over charge that means that this one neutron mass is cut in half therefore we know that the mass has been divided by two if we find that this distance is 0.33 we see that the neutron has now been divided by three and we can read the charge state from the distance between the consecutive isotopes and we can actually calibrate because we have multiple of these measurements we have at least two that is how we can get from a high resolution instrument the charge state of the analyte by taking the distance between the consecutive isotopes so that's a really nice way to figure out this charge and most modern instruments will have isotopic resolution the big exception to this remains the iron trap will not be able to offer it time of flight instruments can do it and we will see later on ordinary traps and free heat transform instruments will also be able to do it so that is why resolution matters and that is why you want it not just because it gives you more accurate mass readings but also because it allows you to determine the charge state and remember for electrospray we really need that piece of information so with that we're done talking about analyzes and our next lecture will be about the detectors thank you very much and see you soon