Transcript: The Basics of Spectrometry-Mass Analyzers Richard Vachet Transcribed by Emily Ott, MA, NIC Master, SC:L, Sr. Interpreter & Transcriber Office of Student Life Disability Services, The Ohio State University

Hello. So the goal of this video lecture is to talk a little bit about as a technique with a specific emphasis on mass analyzers, um, as really the heart of mass .

Before I go on to talking about mass analyzers and specifics, I thought I would just give a general overview about mass spectrometry as a technique, to say something about how it works, and what it's used for. And so mass spectrometry is really a measurement tool, in its simplest form, that's able to provide multiple levels of information about analytes, or compounds of interest that we might be interested in measuring or analyzing.

And really, these three levels that are commonly obtained by mass spectrometry are: 1. molecular weight 2. elemental composition 3. structural information

Now, molecular weight is perhaps the simplest of them to understand. When we're talking about a "mass" , because it can be linked to mass quite easily. Molecular weight by essentially adding up the masses of individual atoms that comprise a particular molecule. Molecular weight, though, is very characteristic of these molecules and therefore a very valuable piece of information because of course all matter has mass, and therefore in principle, all molecules can be measured by mass spectrometry.

But we can also get elemental composition with mass spectrometers by recognizing the fact that atoms that comprise molecules are made up of different atoms that have different isotopes to them. The most common molecules that we like to measure often are things that have carbons, nitrogens, hydrogens in them, and carbon, hydrogen, and nitrogen have isotopes. These have different masses to them, they have different natural abundances as well, so when we measure a molecule that's comprised of different atoms and their different isotopes, that can result in a pattern that is reflected in the mass that we measure for the molecule, and we can use that to determine, often, the number of that atom, or the type of the atom as well, and therefore get empirical formula information and elemental composition about a particular molecule.

Now, structural information is also something that mass spectrometry has more recently become quite good at getting, and typically this is obtained during a experiment or doing MSMS, and in an MSMS experiment, typically what is done is the molecule of interest is brought into the mass spectrometer, and as we'll see, it's ionized, but then we typically break it apart into pieces and measure the masses of the individual pieces, and sort of like a puzzle, piece that back together to get structural information.

Now, in future video series, we'll talk more in detail about tandem mass spectrometry, but we're going to focus today really on the measurement of the masses of the molecules we're interested in.

Now, mass spectrometers are made up of several components, and this is a convenient way to discuss how mass spectrometers work, but the key components of a mass spectrometer are an inlet system that we can use to bring the sample of interest into, and then we can make ions of that sample, and this is a critical part about mass spectrometry that we'll talk a little bit more about in a second, where we can make ions that then are passed to a mass analyzer, where they can be separated according to mass to charge ratio, we'll see, and then detected to provide a .

Now, the mass spectrum is typically shown here as ion abundance, or sometimes ion intensity, as a function of mass to charge ratio, and of course mass to charge ratio is where we're gonna get the information about mass of the compounds that we are analyzing.

Now, you'll see that I draw these components of a mass spectrometer in a vacuum system, in this cartoon, and that is because when we do mass spectrometry, in order to do it best, we do it inside a vacuum system. The reason for that is to keep the pressure very low and minimize interactions that ions have with other neutral molecules or atoms, and this is because ions can be very reactive. As we move ions from one component to another, say from the to the mass analyzer, we want to avoid those ions bumping into things that could compromise their detection.

But, we can actually do mass spectrometry in a way where we can make the ions not under a vacuum but at atmospheric pressure, or at another pressure, and then bring those ions into a vacuum system where they are mass analyzed and detected. Now, as I mentioned, the mass analyzer works best in a vacuum system, and so almost solely, in fact in all commercial mass spectrometers, the mass analysis is done under low pressure to be done optimally.

So just to recap up to this point, what we do when we do mass spectrometry is we are making ions some way, and then we are analyzing them by the mass analyzer. So the key part of this, it's a key part of the whole experiment, is that we need to do some chemistry to make gas phase ions. Ions that eventually go into the mass analyzer in the vacuum system as ions.

Now because we're making ions and measuring their mass, we're actually measuring mass to charge. And so mass spectrometry itself is a little bit of a misnomer. A better name is perhaps mass to charge spectrometry, but that's a mouthful and so we'll stick with mass spectrometry.

Another important thing to just recap and remind you is we're doing this at low pressures, and one of the consequences of measuring ions at low pressures is that if we can do it well, we can actually bring ions to a mass analyzer, to a detector in a very efficient fashion, and if we can also move ions from where they're made to the mass analyzer to detector, we can measure compounds very sensitively, and in fact that is true about modern mass spectrometers. They are very sensitive, can measure very low levels of compounds of interest, and that is one thing that makes them so interesting and powerful.

Now to dig in a little bit more deeply and understand mass spectrometry as a technique in greater depth, it's convenient to think about the three main components of a mass spectrometer: the ion source, mass analyzer, and detector.

And this particular type of presentation, I'm gonna emphasize mostly the mass analyzer, and different ways that we are able to analyze and separate ions according to their mass to charge ratio so that we can detect them, but before I do that, I just want to say a few brief words about how an ion source works, and some of the basic types of ion sources.

So we can think about ion sources and categorize them into two different types. So-called gas phase techniques, and what I will call desorption techniques. Now these gas phase techniques are ones in which we work with compounds that are naturally volatile and thermally stable. And so this ionization by these gas phase techniques involves first volatilizing the compound of interest, and then ionizing it. Usually this is by or , and involves often shooting electrons at the sample to knock off other electrons, um, and then making them ions, and then they're passed on to the mass analyzer.

But there have been more recently developed desorption ionization techniques, such as , and Matrix Assisted Laser Disruption Ionization, or MALDI, that allow us to ionize things that aren't necessarily very volatile, in fact can be non-volatile and sometimes even thermally labile. And in fact this is a very powerful way to make ions of a wider range of molecules of interest because at the end of the day, there are more, it turns out, probably compounds on this planet that are non-volatile and thermally labile than there are ones that are volatile and thermally stable. And so electrospray ionization and MALDI have been very powerful tools for advancing mass spectrometry and being able to apply it to a wider range of types of molecules, for example, biomedical systems or other complicated molecules.

And in fact these techniques of electrospray and MALDI were so important that they, the people who developed them, were awarded with Nobel Prizes back in 2002, John Fenn for electrospray ionization and Koichi Tanaka for his developments in MALDI.

Now detectors I want to say also something briefly about. Detectors, the basic goal of a detector is to take the ions that have been separated by the mass analyzer and convert those ions into some electrical signal, into essentially electrons that can be processed as a current and then a voltage by some computer. And this is done in a variety of different ways, but there are two typical ways that this is done. The first is to use um something that takes ions directly and converts them into electrons and furthermore cascade of electrons that multiplies the signal and amplifies the signal, and this is done with things like electron multipliers or micro channel plates. And again, the idea is it converts ion signal into a current, and this current is achieved by the electrons that are produced from the initial ion hitting some surface.

We can also detect ions in another way using image detection, where here, this is a more non-destructive approach to detecting ions, where now ions induce a current on nearby plates as they oscillate near a plate, and this resulting image current peak can be used to determine um, essentially that the ions are there, and to some degree how much of those ions are here. An interesting feature of the image detection is that it is non-destructive compared to using an or a micro channel plate.

Alright, so now I'm gonna turn to the bulk of the rest of my presentation, and that is to talk about mass analyzers themselves. Now, when we talk about mass analyzers, it turns out there are many different types of mass analyzers that we can talk about, from time-of-flight mass analyzers to double focusing mass analyzers, quadrupoles and so on, and there are many ways to couple these different types of mass analyzers together to make so-called hybrid mass analyzers that combine one type of mass analyzer with another.

Now, I'm not gonna be able to go through the details of all these different types of mass analyzers, but I thought I would tell you that even though we have many different types of mass analyzers, they all work on the same basic principle, and that is Newton's second law, or F=ma. The idea here is that we use either a magnetic and/or an electric field to exert a force on an ion. This force causes ions to accelerate to different amounts that ultimately depends on the mass of the ion. And so this basic principle of F=ma, really, if you understand this well enough, you can understand how most mass analyzers work.

Now, if you're thinking about this, nowhere in this equation does charge show up, and in fact charge does show up in the "force" part of this equation, and you can understand that by thinking about the fact that a given electric field will act on an ion with two charges to twice an extent as an ion with a single charge, and so the charge shows up in this force; the force acts greater on more highly charged ions. But at the end of the day, that force causes different masses to accelerate to different degrees, and therefore the basis of a mass analyzer.

Now, so, basic idea, a mass analyzer works by separating ions according to their mass to charge ratios, and physics is involved.

Before I tell you the details about some of these particular mass analyzers, I thought I would introduce some terminology to help us understand. These are terms that are often use to characterize how good the mass measurement has been done, and really one figure of merit for describing how a particular mass analyzer is. And these two terms are "mass accuracy" and "mass resolution," or we'll see with the second, "resolving power" often is a more common term used to describe how well a mass analyzer is doing at resolution.

So first, "mass accuracy." So mass accuracy simply is how close to the true mass to charge ratio is an ion that is measured? Now we can think about this in the relative sense, or in an absolute sense. In a relative sense, we typically quantify this in terms of parts per million, and it's simply the measured mass and its difference from the theoretical mass, divided by what theoretically should be times ten to the sixth the get the parts per million. And so this is a relative measure and often a way to characterize how good a mass analysis has been, and in mass spectrometers, mass accuracy in these terms often can be sub-parts per million, so parts per billion mass accuracy all the way up to tens to hundreds of parts per million, and this will depend on the type of mass analyzer and how careful the measurement is made.

We can also think about an absolute measure for mass accuracy as well, and we often think about this in terms of millimass units, or perhaps millidaltons, and what we're talking about is really the number of decimal points that we can get in the measurement.

Mass resolution is how well we are able to separate two ions that have very similar mass to charge ratios. How well we can resolve one ion that has a similar mass to charge ratio as another. Now we'll often quantify this using resolving power, and resolving power, we think about doing an equation where we look at the mass of interest, and look at delta mass, and delta M here can take two real definitions. The first is the more common one; we look at the width of a given peak in a mass spectrum at its half maximum, and that often is most commonly used as delta M in this equation for resolving power. But we also can think about delta M as the mass to charge difference between two peaks in a spectrum that are resolved.

Just to give you an example of what I mean by mass resolution, I show a spectrum here that has relatively low resolution; the mass analyzer used here has a mass resolving power that is relatively low for modern mass spectrometers, and so this is a measurement of a peptide, and this peptide has two charges on it, and what we can see is that we can resolve two peaks that are nearby, but barely. And you can imagine if we had resolving power that was lower than this 1850, the two peaks that are near one another could not be resolved. Clearly, if we go up higher though, we can more clearly separate the two nearby mass to charge ratios with a higher resolving power, and many mass analyzers can even go higher than this, and in fact we can get mass resolutions with resolving powers for different mass analyzers that can range from thousands all the way to millions, depending on the mass analyzers, and I will talk more about that in a second.

Another reason I showed the spectrum is I want to distinguish between mass resolution and mass accuracy. And I did this by, in this case we see a pretty decent resolution for this particular ion that we can separate two nearby peaks from one another, two nearby mass to charge ratios, but it turns out in this case, and I purposely did this, we actually have a poor mass accuracy. Now this ion that was measured was measured here with a mass to charge ratio of something slightly below 649. But the true mass of this particular ion that we measured is a little bit higher than that, and this difference from the theoretical, which is shown by the dotted line, and what we measure, emphasizes the mass accuracy in this case, which is relatively poor and was purposely done so that you could see it, but what I also want to distinguish is that getting a much better resolution in this case would not necessarily lead to a better mass accuracy, that those are two separate figures of merit.

So now let me talk a little bit about the details of mass analyzers, and we can categorize mass analyzers into two different types. The first are beam type mass analyzers, and these are ones where the ions are accelerated through an electric or magnetic field and are separated then either in time or space to arrive at a mass spectrum, and they hit the detector after they have been separated.

We also can mass analyze a collection of ions by trapping them in an electric field or a magnetic field, and these ions then will oscillate in a mass-dependent way, and we can either eject them out of the trapping field to detect them, or in other cases we can detect them while they are in the trapping field itself as a way to arrive at a mass spectrum.

So I'm gonna break down these and talk about these two different classes of mass analyzers.

So let's talk first about the beam type mass analyzers. So really there are three primary types of beam type mass analyzers. The time-of-flight mass analyzer, or TOF, a double-focusing mass analyzer which is sometimes called a sector mass analyzer, and a quadrupole mass analyzer which is sometimes called a quadrupole mass filter.

Now each of these really on the principle of ions being brought through an electric or magnetic field and separated according to mass to charge ratio and then detected. Now I'm only gonna talk about the time-of-flight mass analyzer and a quadrupole mass analyzer, because double-focusing mass analyzers are used much less commonly these days. Historically, double-focusing mass spectrometers were used quite a lot, especially for high mass accuracy measurements and to study ion energetics, but now they're used much less commonly.

So let me first talk about the time-of-flight mass analyzer. So a time-of-flight mass analyzer, the name itself pretty much tells you how the mass analysis is done. We look at how long it takes for an ion, after it's made, to transverse from where it's made to the detector. And so the time it takes to fly from one location to the other ultimately is going to be related to its mass to charge ratio.

Now it's related that way because what we do is when we make ions, we then accelerate all the ions that are made with the same kinetic energy down a flight tube of a given length, often half a meter to two meters. The time that it takes then of course, it's like a race. The ions from the beginning to the end, how long they take will depend upon the velocity that they're moving at, and that velocity ultimately will depend on their mass to charge ratio when we think about this kinetic energy equation here. When ions are all accelerated at the same kinetic energy, they will have different velocities that are dependent on what their mass to charge ratio is. And this serves as the basis of how time-of-flight can acquire a mass spectrum. The time it takes can be seen by this equation, and we can see that it's related to the mass to charge ratio of an ion. Typically it's the case if ions of a given charge are accelerated to a given kinetic energy, they give a velocity that's mass to charge dependent, and you can see from this equation that the less massive ions or the lower mass to charge ratios are gonna reach the detector before the more massive, or the higher mass to charge ratio ions.

Now just a couple things about the time-of-flight mass analyzer, to put it in the context of mass accuracy and resolving power, modern time-of-flight mass analyzers have what I would say moderate mass accuracy, moderate to high mass accuracy, and moderate to high resolving power. And historically, time-of-flights did not have very good resolving power, but some recent advances in terms of how the measurement's made, how the voltages are applied, um, has led to better resolving powers and turned into a really high performance type of mass analyzer.

Now one interesting feature of time-of-flights that's worth pointing out is that they have an unlimited mass to charge range in principle. We can measure, in principle, as long as we're willing to wait long enough, any mass to change ratio. There are complications that make that, in practice, not possible, but in principle, unlimited mass to charge range is achievable with a time-of-flight mass analyzer.

The second type of beam-type analyzer that we want to talk about is a quadrupole mass filter. Now this picture here, I show a quadrupole mass filter, and as the name suggests, quadrupole has four poles associated with it, or four rods. In this picture, I show only three of them just so you can see more clearly how the ions might move through a quadrupole mass filter, but the way a quadrupole mass analyzer works is that we apply an RF and DC voltages to these rods, adjacent rods will have RF voltages that are 180 degrees out of phase, for example this rod and this rod would have an RF voltage 180 degrees out of phase; they would also have DC voltages of opposite polarity applied to them. The result, when we do this, is that we generate an electrodynamic field that influences the motion of the ions as they go from the ion source to the detector in two dimensions.

So the ions are influenced up and down in this picture, and in and out of the screen by the electrodynamic field. The electrodynamic field itself, to a first approximation, does not affect an ion's motion as it transverse down the axis from the ion source to the detector; it only affects it in two dimensions. Now this quadrupole mass filter is able to selectively pass through a very small range of mass to charge ratios depending upon the RF amplitude and DC amplitudes applied to the rods. Now we can acquire a mass spectrum by increasing those RF and DC amplitudes done at a constant ratio between the RF and the DC, and then we can allow smaller, then bigger, and then the biggest ions to go through the mass analyzer to the detector.

Now when we do this, the mass analyzer itself has, compared to at least the time-of-flight, relatively low mass accuracy and resolving power. Now, we have some degree of control of the resolving power, but it's relatively low. One feature of the quadrupole mass filter or mass analyzer that's really powerful is that it's really excellent at doing quantitation, and it's commonly used for that purpose. We also can use a quadrupole if we link together more than one quadrupole, often three of them together to make a triple quadrupole, we can also do tandem mass spectrometry quite well.

So now I turn my attention to talking about the trapping analyzers. And the trapping analyzers really, there are three primary types to them, and they're shown here in cartoon form. One is a quadrupole , another is a Fourier transform ion cyclotron resonance, and the third is an .

Now in these devices, as I mentioned a few slides ago, the mass analysis is done by bringing ions into these traps, and a range of ions can be trapped, and then either selectively ejected out to do a mass analysis, or the ions, their masses are determined while they still are trapped.

So let me first talk about the . And the quadrupole ion trap really comes in two types. And I'll talk first about what's known or what we refer to as a three dimensional quadrupole ion trap. And a three dimensional quadrupole ion trap, unlike the quadrupole mass filter where the "quad" suggested four rods, in this case, the quadrupole ion trap has three electrodes. We have a ring electrode shown here in a cutaway view. This ring electrode forms sort of like a donut, and sandwiched up against this donut of a ring electrode are two end cap electrodes that are sort of like doorknobs. And this ring electrode and the end cap electrodes provide the space where the ions can be trapped.

Now the ions themselves are trapped by the application of an RF voltage to the ring electrode. This ring electrode relative to the end cap electrodes that are grounded generate a three dimensional trapping field that's actually a quadrupolar field. This three dimensional trapping field allows us, depending on the amplitude of the RF that we've applied to the ring electrode allows us to trap a wide range of mass to charge ratios; in fact all the ions above a certain mass to charge ratio can be trapped.

Now to turn this quadrupole ion trap into a mass analyzer, we mass selectively make the ions that we've trapped unstable. And we do this by increasing the RF amplitude applied to the ring electrode in such a way that we selectively send out small, then medium size, then larger ions to hit a detector. And then that way, um, acquire a mass spectrum.

Now the quadrupole ion trap mass analyzer is much like the quadrupole in terms of its figures of merit. It has a relatively low mass accuracy and relatively low resolving power, although like in the quadrupole mass filter we can vary the resolving power by changing certain parameters quite readily. But one of the key features of a quadrupole ion trap is that it's exceedingly excellent at doing tandem mass spectrometry, or MSMS. And in fact it can do multiple stages of MSMS, or "MS to the end" as we refer to it. And so it has great power in that regard.

Now, an analogous type of quadrupole ion trap is what we call a 2D or a two dimensional quadrupole ion trap, and this is based on the same principle of quadrupole mass filter in that we have four rods, and we're applying an RF voltage to these rods, but in addition, we apply to the ends in this particular configuration a DC voltage that maintains the ions trapped along this what I will call "axial" dimension. The RF voltage applied to the rods traps the ions in the 2 dimensions, and this DC voltage applied between these two ends traps it in the third dimension, which allows us to provide this quadrupole ion trap. Now much like the quadrupole mass analyzer, the rods in this particular case, alternating rods have RF amplitudes that are 180 degrees out of phase with regard to one another, as can be seen here in these equations. The X rods would be in the same phase, but the Y rods would have a different phase associated with them.

Now much like the three dimensional mass analyzer, or quadrupole ion trap mass analyzer, the way we acquire a mass spectrum here is to mass selectively untrap or mass selectively make the ions unstable, send them out of the field to be detected. And we do this in much the same way, we increase RF amplitude in the small, then the medium size, then the bigger ions are ejected out to be detected. Now much like the three dimensional ion trap, relatively low mass accuracy and resolving power, but also quite good at doing tandem mass spectrometry.

Another type of trapping mass analyzer is a ion cyclotron resonance, or a Fourier transform ion cyclotron resonance. In this case, we use a magnetic field, predominately, to trap the ions. Um, and often these magnetic fields are very strong magnetic fields, something on the order of 7 tesla to maybe even 15 tesla, magnets that are often the size of NMR magnets, and this magnetic field actually causes trapping of the ions in two dimensions. We then also have to use an electric field to provide the trapping of a third dimension, to maintain the ions into this trap.

Now as ions are trapped in this magnetic field, they will oscillate around the magnetic field with frequencies that are dependent upon their mass to charge ratio, and in fact the frequencies at which they oscillate are inversely proportional to their mass to charge ratio, and so ions that are lower mass to charge ratios oscillate with higher frequencies. The larger mass to charge ratios oscillate with lower frequencies.

Now as these ions oscillate in the field with their particular frequency, they can then be detected non-destructively, and we do this by having them pass near plates that pick up an image current. As these ions go near particular plates, the current that is induced on these plates can be measured, amplified, and ultimately be a record of the frequency of the ions that are trapped in this cell. Now because many ions can be trapped at once, this leads to multiple frequencies, and therefore destructive and constructive interference; we then, therefore have to take a Fourier transform of the signal that results to convert it into the frequency information that allows us then to convert it into a mass spectrum. Fourier transform ion cyclotron resonances, if we look at the figures of merit for them, they have very high mass accuracy and very high resolving power compared to the time-of-flight, quadrupole mass analyzers, and the ion traps, they have much much higher mass accuracies and higher resolving powers associated with them. Because it is a trapping device, one of the things also they can do is we can perform tandem mass spectrometry experiments in a Fourier transform ion cyclotron resonance, although commonly the ability to do MSMS is not as efficient as in the quadrupole ion trap mass analyzers.

Now finally, the final mass analyzer that I want to say something about is an orbitrap mass analyzer. Of all the ones that I talked about today, this is the one that has been most recently developed and commercialized, and this orbitrap also is a trap, but now it uses an electric field applied to this center electrode. This electric field, relative to the shielding electrodes around this center electrode, allow ions to be brought in, and they can oscillate around this center electrode, and remain trapped. Now these oscillations around these center electrodes also are combined with oscillations back and forth along this z axis here, and these oscillations along the z axis, the frequency at which those oscillations occur are mass to charge dependent. Again, much like in the ion cyclotron resonance, there's an inverse proportionality to the frequency and mass to charge ratios, so smaller mass to charge ratios oscillate at higher frequencies than larger mass to charge ratios.

During these oscillations, much like with the FTICR, we can non-destructively detect these ions by measuring an image current, again, on some detection electrodes, and this allows for, again, the collection of the constructive and destructive interference associated with multiple ions and multiple frequencies oscillating back and forth. We can use then a Fourier transform to deconvolute that resulting time domain signal into a frequency domain that can then be related to mass to charge ratio.

And when we do this, this has many of the features like an FTICR in that we can get very high mass accuracies and resolving powers, um, one thing that this particular trapping mass analyzer is un-capable of doing at least currently is being able to do tandem mass spectrometry. Um, that's not possible in this orbitrap, and so it is for mass detection alone right now.

Now one final thing to mention, as I alluded to many slides ago, often times many mass analyzers can be coupled together to really reap the benefits of different mass analyzers. And I saw one very specific example of this where we have a coupling together of a two dimensional quadrupole ion trap together with an orbitrap mass analyzer. Now the coupling of these two together allows us in the same particular instrument reap the benefits and the advantages of a quadrupole ion trap, which are most notably the ability to do tandem mass spectrometry very well, and also to do multiple stages of mass spectrometry, together with the orbitrap, which cannot do MSMS, but has very high mass accuracy and high resolving power that the quadrupole ion trap itself does not. And so by doing these two in the same instrument, we can reap the benefits of the two that we couldn't achieve separately.

Now, just to summarize what I've said during this lecture, mass spectrometry ultimately relies on making ions of a sample, and then analyzing those ions using a mass analyzer, and then passing them along to the detector. And because we're making ions, we're measuring their mass to charge ratio, we could refer to this as mass to charge spectrometry, although that's a mouthful and we typically call it mass spectrometry.

These mass analyzers ultimately are working on the basic principle of F=ma. Newton's second law. And so it's a simple physics experiment that we separate ions according to mass to charge ratios, and we can do this in a variety of different ways, and this particular lecture, I mentioned two general types, beam type mass analyzers and trapping type mass analyzers, and I also talked about the different advantages and disadvantages that particular mass analyzers have. And very recently, something that's been done commercially is that combining two different mass analyzers together allows us to reap the benefits that individual mass analyzers have themselves without necessarily having the disadvantages associated with them.

So there ends my brief description of mass analyzers as they're used in mass spectrometry.