6 Mass Spectrometry 6.1 How does mass spectrometry work? This is a technique that allows us to measure the molecular weight of the molecule. Additionally we can also gain valuable information about unknown substances by measuring the masses of the fragments produced when high energy molecules fly apart. You will notice the different term used in the title: spectrometry, not spectroscopy. This is because there is no absorption or emission of radiation involved. The spectrum is a graph of particle mass (horizontal axis) against number of particles (vertical axis). A very small amount of sample is introduced into the mass spectrometer and bombarded by a beam of high energy, most often in the form of rapidly moving electrons. When this high energy beam strikes the organic molecule it dislodges an outer shell electron and produces a positively charged species, as shown in Equation 1. This ionised molecule is known as the molecular ion, and has the same mass as the neutral compound. In addition to causing ionization, electron bombardment transfers such a large amount of energy to the target molecules that they often fly apart into a number of smaller pieces, some retaining the positive charge and some remaining neutral. All the positive ions of the various masses are separated from other particles and then by their different masses to produce the spectrum. The peaks corresponding to the break up of the molecule are called the fragmentation pattern. M electron beam F1+
M+
F2+ F3 FIGURE 6.1 Ionisation and fragmentation process A typical mass spectrum is shown in Figure 6.2. fragmentation
pattern base peak
135
77
intensity (%) 104
165
180
molecular ion FIGURE 6.2 A typical mass spectrum m/e (mass to charge ratio)
6. Mass Spectrometry 6.2 Interpreting mass spectra 1. Molecular weight – in most, but not all cases, some of the molecular ion remains unfragmented and reaches the detection system. Therefore, it produces a peak on the spectrum. Therefore, the highest mass peak in the spectrum is most likely to be that of the molecular ion, and therefore, equal to the molecular weight of the compound. For example, the highest mass peak in the spectrum in Figure 1.17 is 180, and could therefore be the molecular weight of the compound. How can you be sure that it is the molecular ion? This is not simply answered, but when we look at the instrumentation side of mass spectrometry, some clues will be gained. One point to make – in most spectra, you will notice a very small peak one higher than what is described as the molecular ion peak. This is due to the small natural proportion of carbon‐13 atoms in the carbon population. 1 in 100 carbon atoms weighs 13, not 12, so any molecule with this atom in it will weigh one more than the majority. It does not alter the molecular ion value. 2. Molecular fingerprint – each molecule will always fragment in the same way, so compounds can be positively identified by a comparison of their mass spectra in the same fashion as we do with infrared. However, the mass spectra of very similar compounds are very similar, and not as good a “fingerprint” as the infrared spectra. This is shown in Figure 6.3. FIGURE 6.3 Mass spectra of (a) 1,3‐dimethylbenzene and (b) 1,4‐dimethylbenzene 3. Isotope peaks – as mass spectrometry measures individual ions, it shows a peak for each isotope of a mixture. Hence in chlorine for example, we see the peaks due to both the 37Cl and 35Cl and these always occur in the natural 3:1 ratio. These are referred to as isotope peaks. Bromine shows peaks for 79Br and 81Br, and these are often observed as repeating peaks in their 1:1 isotopic ratios throughout the mass spectrum. These are shown in Figure 6.4. 4. The “nitrogen rule” – a molecule of even number molecular weight must contain either no nitrogen or an even number of nitrogen; likewise a molecular weight which is odd signifies an odd number of nitrogen. It doesn’t matter what other elements are in the compound. It isn’t easy to prove this, but you will not be able to find an example that disproves it! 5. Characteristic fragmentation losses – some functional groups fragment in a particular and consistent way; for example, ethyl esters always lose the OCH2CH3 group, thus showing a strong peak which has a mass loss of 45 less than the molecular ion. This is shown in Figure 6.5. AIT
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6. Mass Spectrometry Peaks showing presence of Cl 3:1, 2 apart Peaks showing presence of Br 3:1, 2 apart FIGURE 6.4 Mass spectra of (a) 2‐chlorobenzoic acid and (b) 2‐bromobenzoic acid Loss of 45 = Loss of OCH2CH3 105 <= 150
99 <= 144 FIGURE 6.5 Mass spectra of (a) ethyl benzoate and (b) ethyl hexanoate CLASS EXERCISE 6.1 What loss would expect to see from the molecular ion if the compound was a methyl ester? AIT
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6. Mass Spectrometry CLASS EXERCISE 6.2 What information can you obtain from the mass spectra below? AIT
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6. Mass Spectrometry 6.3 Instrumental components Mass spectrometers, whether they are standalone instruments or detectors for other instruments (eg GC, HPLC, ICP) have a common set of components, as shown in Figure 6.6. There are different specific types of each component, which we will look at in this chapter. high vacuum
Sample
Inlet
Fragmentation
Chamber
Mass
Analyser
Detector
FIGURE 6.6 Schematic diagram of a mass spectrometer Sample inlet A very small quantity of sample (10‐100 g) is required in the mass spectrometer, and it is also necessary that the sample is carried into the high vacuum within the instrument without leaks. For substances with relatively high volatility (liquids, gases and some solids), the sample is introduced into a heated injection port at 300C, very much like one in a GC. The material vaporises and passes into the fragmentation chamber. If the material is not volatile or is unstable when heated, then a direct injection method is used, where the sample is held in a tiny cup, and inserted directly into the fragmentation chamber. Fragmentation chamber (ion source) Mass spectrometry relies on the detection of charged fragments of the original molecules. Therefore, some means of ionising the sample is required. There are a number of methods for achieving this, including: 
electron impact 
chemical ionisation 
fast atom bombardment The most common by far is electron impact, where a beam of high speed electrons comes into contact with the sample. The degree to which fragmentation occurs depends partly on the compound, but also on the energy of the electron beam. The normal electron energy is 70eV, which means the voltage difference in the electric field is 70V. In some spectra, no molecular ion appears, which makes identification difficult. This can be avoided by lowering the voltage, causing less energy to be transferred to the molecule, which will be less unstable. Vacuum The vacuum inside a mass spectrometer is very substantial: around 10‐7 torr (1 atm is 760 torr). This requires two levels of vacuum pump: one to remove the bulk of the air, and the ultra‐high vacuum pump to remove the last traces. CLASS EXERCISE 6.3 Why is such a high vacuum required? AIT
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6. Mass Spectrometry Mass analyser The mass analyser is the equivalent of the monochromator in a spectrophotometer. It separates the fragments on the basis of their different masses. Before the mass analyser is an ion filter, which blocks any non‐positive fragment from passing into the mass analyser. It does this by employing a series of negatively charged plates, with increasing voltages. These attract the positive ions from the sample, but repel any negatives and do not affect the neutral fragments. The vacuum system draws the non‐positive fragments away. The positive ions are not lost at the plates because each plate has a gap in it, and when an ion is drawn towards the first one, it feels the attraction of the second and moves through the gap to it, and so on, until it bursts through the gap in the last plate because it moving so fast. It then enters the mass analyser. Figure 6.2 shows the ion filter. ‐ ‐‐ ‐‐‐
F+
F-
FIGURE 6.7 A typical ion filter Mass analysers are of a number of different types, differing in the way that they separate the different mass particles: 
magnetic sector 
quadrupole 
time‐of‐flight The magnetic sector mass analyser was the first to be used in mass spectrometers. It uses a large magnetic with a curved path bored into its centre. The particles move into the magnetic field and their path bends at a radius proportional to their mass: the heavier the mass, the wider the curve of its path (see Figure 6.8). Think of a motor bike and a semitrailer going around a bend at the same speed: the motor bike can take a tighter bend. The radius of the path is fixed so only one mass can pass through at a time: the semitrailer can’t make the bend, and crashes into the wall. To allow different masses through, the magnetic field must be changed (like in the NMR scan). Thus, at each magnetic field strength, a different mass particle can move through to the detector. The problem with magnetic sector analysers is the size of the magnet and the relatively slow scan speed possible. The quadrupole mass analyser works very differently, and reduces both of these problems, so we have a compact (briefcase‐sized), rapid‐scan (less than 1 second) mass analyser. AIT
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6. Mass Spectrometry magnetic field particle with wrong mass for curve curved path particle with correct mass for curve FIGURE 6.8 A magnetic sector mass analyser CLASS EXERCISE 6.4 Watch the video and answer the following questions. (a) Why is the quadrupole analyser so called? Draw a diagram showing its basic shape. (b) How does it separate the different mass particles? Each of these mass analysers has a different resolution capability: the ability to tell similar but not identical masses apart. A magnetic sector analyser can typically distinguish masses 0.05 mass units apart, whereas for a quadrupole, it is 0.1. Because of its speed, small size and lower cost, the quadrupole analyser finds its main use as the analyser in an interfaced instrument, where the MS is serving as a detector for another instrument. These are discussed in the next section. AIT
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6. Mass Spectrometry Detectors Back in the old days, sensitised paper (special film in other words) was the means of detection. After the “Stone Age”, detectors related to photomultiplier tubes in that an incoming charged particle (rather than a photon) liberates an electron from the point of contact, and this electron causes a cascade of electrons inside the detector, thus generating a measurable current. 6.4 The MS as a detector It is increasingly common for two different instruments to be linked together, so that the second becomes the detector for the first. These are known commonly as hyphenated instruments. The most common example of this is the mass spectrometer detector for GC, HPLC and ICP, giving the acronyms GS‐MS, HPLC‐MS and ICP‐MS. In each case, the great advantage of the MS as a detector is its ability not just to detect the analyte species, but to produce a characteristic response (i.e. a fingerprint or a formula weight). The hard part is the plumbing that connects the two: the outlet from the first instrument is the sample inlet for the mass spectrometer. GC‐MS This was the first hyphenated instrument, and remains the most common. It is one of the standard techniques for drug and pesticide testing. How is it connected to the gas chromatograph? The amount of material required in a mass spectrometer is very small – micrograms – which is the level of analyte coming from the GC column. However, what also comes from the GC column is a large amount of carrier gas, which would swamp the MS. There are various mechanisms to remove the bulk of the helium, the simplest relying on the larger mass (and therefore momentum) of the analyte molecules, compared to the helium carrier gas. A vacuum pump is placed in the interface between column and MS inlet, and it pulls the light helium gases into it, while the heavier analyte molecules move into the MS, as shown in Figure 6.9. MS inlet
GC column
vacuum pump
FIGURE 6.9 Typical GC/MS interface How does the MSD produce a chromatogram? In a normal chromatographic run, the mass spectrometer produces a spectrum of the fragment masses a number of times each second. What we want in a chromatogram is not a lot of mass spectra, however, but rather a graph of concentration of detected species against time. To produce this from the mass spectra, the detection system also adds up all the fragments of any mass making up each spectrum. This value is plotted against time, and is known as the total ion current (TIC), because each fragment that is detected in the MS is a charged particle. So the chromatogram is a series of points, each linked to a mass spectrum. The software controlling the GC‐MS allows access to the mass spectra, so we can not only see the amount of a particular AIT
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6. Mass Spectrometry species at a particular time (from the peak area), but also get a fingerprint of the compound causing the peak. Most software also come with a comprehensive database of mass spectra, which are electronically compared to the sample spectrum, and the best matches listed. So we have a GC detector that is able to identify the compound causing the peaks for us! How can the MSD be made selective? In TIC mode, a MSD is a universal detector, but the method of detection in the MS lowers the sensitivity. This is because so many of the particles never reach the detector, crashing into the rods of the quadrupole because their mass isn’t the one allowed at the moment. However, if you are interested in one particular species, then you can change the scan mode of the MS to single ion monitoring (SIM), which sets the detector to only pick up fragments of a specified mass – one that is characteristic of the species of interest. The mass spectrum facility is no longer available, but that doesn’t matter because you already know what you are interested in analysing. This mode has two major advantages: 
the sensitivity is greatly increased 
interfering peaks of no interest do not appear on the chromatogram, even if they emerge from the GC column at the same time as the analyte How do MSDs compare to other GC detectors? They are far more expensive to buy and the startup/shutdown procedures are more complex and time‐consuming. Running in TIC mode, MSDs are a little less sensitive than flame ionisation detectors and electron capture detectors, but have the great advantage of identification and are also universal. In SIM mode, they are selective and as sensitive as any other detector. HPLC‐MS The interfacing of an HPLC to a mass spectrometer followed after the successful connection of GC to MS, with all the advantages that the MS detector brings. The same advantages would be available to a HPLC‐MS instrument. The problem of the mobile phase swamping the mass spectrometer inlet was encountered with GC‐
MS. The problem is far more serious with HPLC where the mobile phase is, of course, a liquid. This makes it much harder to remove than the small helium atoms in GC. The MS inlet can cope with microlitres per minute of material, while the usual flow rate from an HPLC column is millilitres per minute. At least twenty years of research and development went into solving the problem of removing the solvent without destroying the analytes. Rapid heating was the first approach tried, but this could not be made to work consistently with all analytes and mobile phases. The current solution is known as electrospray ionisation, and combines two stages in the process: removal of solvent and analyte ionisation (see Figure 6.10). The eluent from the HPLC column is sprayed through a nebuliser into a chamber at atmospheric pressure in the presence of a strong electrostatic field and heated drying gas. The electrostatic field causes further dissociation of the analyte molecules. The heated drying gas causes the solvent in the droplets to evaporate. As the droplets shrink, the charge concentration in the droplets increases. Eventually, the repulsive force between ions with like charges exceeds the cohesive forces and ions are ejected (desorbed) into the gas phase. These ions are attracted to and pass through a capillary into the mass analyser. AIT
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6. Mass Spectrometry FIGURE 6.10 Electrospray ionisation (from www.agilent.com) Because the process of ionisation has not relied on heat, as it does in conventional MS sample inlets, non‐volatile or heat‐unstable compounds can be readily analysed by this technique. The MS detector then functions in the same way as discussed in GC‐MS. Applications of HPLC‐MS are as wide as those for HPLC itself: remember that the MS is no more than just another detector. However, the three most important areas of use for this technique are: 
pharmaceuticals – particularly the analysis of illegal drugs (recreational or performance‐
enhancing) 
pesticides – some pesticides are very heat‐unstable, and so are not analysable by GCMS 
biological macromolecules – proteins and nucleotides are biologically important molecules with very high FWs ICP‐MS Plasmas generate atomic vapours, including ions, while mass spectrometers analyse ions. Therefore, the interfacing of the two seems appropriate. You can either see the plasma as the ion source for the mass spectrometer, or the MS as the detector for the ICP. Whichever, the result is the most sensitive instrument currently available for atomic analysis. It is also the most expensive at something approaching $500, 000. What does all this extra cost bring? Firstly, the number of ions generated in the plasma far exceeds the number of photons, so the sensitivity of ICP‐MS is much greater than ICP optical emission spectroscopy. In fact, ICP‐MS is more than 100 times more sensitive than electrothermal AAS, so that ICP‐MS can accurately measure down towards 10 nanograms/L, while still being capable of analysing up to 100 ug/L – a very wide working range. Very few species need to be measured at below 1 ug/L, but the working range of the ICP‐MS means that its limits are not being pushed by a solution of 10 ug/L, unless EAAS, where this is the bottom end of its range. The other important, and almost unique, capability of ICP‐MS is its ability to separate and analyse different isotopes of the same element. This means that natural versus radioactive sources of a particular element can be detected. For example, the four natural isotopes of lead are 204, 206, 207 and 208 at with abundances of 1, 24, 23 and 53% respectively. Any major difference in these abundances means that the lead in the sample must have come from a radioactive source. Because the different isotopes have the same spectral properties, an ICP emission instrument could not tell them apart. AIT
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6. Mass Spectrometry What Yo
ou Need To B
Be Able To D
Do 
draaw a schemaatic diagram showing the
e componentts of a typicaal mass specttrometer 
name and desccribe the opeeration of co
ommon typess 
listt instruments using MS d
detectors 
listt the advantaages of MS d
detectors in h
hyphenated instrumentss 
describe what needs to be achieved in the interfacee between aan MS and th
he other instrrument n Questions Revision
1.
W
Why is mass spectroscopyy an inapprop
priate term? 2.
Giive TWO reasons why a vvery high vaccuum is requ
uired inside aa mass spectrometer. 3.
Ho
ow could you
u adjust the settings of aan MS instrum
ment to redu
uce the amount of fragm
mentation to
o ensure thatt a molecularr ion could b
be observed?? 4.
Giive ONE advaantage of a q
quadrupole m
mass analyseer over its altternatives. 5.
Brriefly describ
be the meanss by which a GC is connected to a MSS. 6.
Exxplain how a MS detector is used to p
produce a ch
hromatogram
m. 7.
W
What is the meaning off the term “single (or selective) ion monitorring”? Und
der what cirrcumstancess would it bee used? 8.
W
What is the main advantagge of a MS detector for aa GC or HPLC
C? 9.
W
What does an ICP‐MS do b
better than ICP emission? 10. W
What can an ICP‐MS do th
hat ICP emisssion cannot?
11. W
What information can you
u obtain from
m the mass sp
pectra below
w? (aa) (b) AIT
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6. Mass Spectrometry Answers to revision questions 1.
No radiation involved 2.
Remove contaminants, increase lifetime of fragments by reducing collisions 3.
Reduce voltage (energy) of electron beam 4.
Resolution 5.
Narrow inlet aligned closely with end of column, light He mobile phase atoms drawn away by vacuum; heavier analyte molecules keep moving straight ahead 6.
The total number of ions (of all masses) in each spectrum is used to create a point on the chromatogram 7.
Detector set to allow only fragments of one mass to pass. Quantitative analysis. 8.
Ability to identify compound causing peak by matching MS to library spectrum 9.
Better sensitivity 10.
Ability to distinguish between isotopes of same element 11.
(a) FW 184/186, presence of Cl, 0 or even number of Ns, loss of 45 suggests ethyl ester (b) FW 113, no Cl or Br, odd number of Ns, no useful losses AIT
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