The University of Bristol, School of Chemistry MASS SPECTROMETRY RESOURCE

Here we list several basic questions regarding mass spectrometry - with their answers below.

1. What is meant by the term 'mass spectrometry'?
2. What is 'ionisation' and why is it needed?
3. What is meant by the term 'analyte'?
4. What is the difference between 'cations' and 'anions'?
5. What is the 'mass-to-charge' ratio scale and what are 'multiply charged' ions?
6. What are meant by the terms 'mass precision', 'mass measurement accuracy' and 'mass resolution'?
7. What is the difference between 'high resolution' and 'low resolution'?
8. What is the difference between 'average mass', 'nominal mass', 'exact mass' and 'accurate-mass'?
9. What are 'isobaric' ions?
10. What are isotopes and how do they affect a mass spectrum?

1. What is meant by the term 'mass spectrometry'?

In its most basic form, mass spectrometry (MS) is an analytical technique for measuring the mass of single chemical (and atomic) species. In the last few decades, MS has been coupled to the chromatographic methods: gas chromatography (GC) and liquid chromatography (LC) to allow the analysis of chemical mixtures. These so called 'hyphenated techniques' of GC-MS and LC-MS are now very common and are to be found as standard techniques in any chemical, biochemical, environmental, forensic and pharmaceutical laboratory. MS instruments have even been taken into space and to Mars! With the development of cheaper multiple sector mass spectrometers, tandem MS (MS/MS) or multistage MS (MSⁿ) are becoming more common. These techniques can be used for structure determination, and in combination with LC-MS (for example), MS/MS is becoming a common place tool for drug discovery and drug metabolism studies.

2. What is 'ionisation' and why is it needed?

Ionisation is a physical process brought about in the source of the mass spectrometer. There are several possible ionisation methods and techniques in common use today - which technique is used depends on the compound being analysed and the type of information required. The theory section of this web site describes the most common techniques.

Strictly speaking you don't need to ionise compounds to analyse them by MS. You could make use of gravity broadening to separate molecules by their mass, but this would require instruments with flight tubes in the kilometre range! There are huge benefits to ionisation. The most obvious of which is that it allows instruments to be built in the laboratory scale - in fact 'benchtop' instruments are very common now. The smallest mass spectrometers actually fit inside a briefcase and just such instruments are used commonly by the military (explosives testing), customs and excise (drugs testing) and the rescue services (testing for toxic fumes in fires). Ionisation also allows the analyst to make use of the basic laws of physics to separate ions according to their mass and charge (mass to charge ratio, m/z) and to perform gas-phase reactions with those ions (see later).

3. What is meant by the term 'analyte'?

Quite simply, the analyte is the compound or species being analysed.

4. What is the difference between 'cations' and 'anions'?

A cation is a positively charged ion. These can be produced either through loss of an electron, by the addition of a cation (e.g. a proton or a sodium cation) or through separation of a salt into its constituent ions.

An anion is a negatively charged ion. These can be produced by gaining an electron, by the addition of an anion or by loss of the cation from a salt.

5. What is the 'mass-to-charge' ratio scale and what are 'multiply charged' ions?

The mass-to-charge ratio (m/z) of an ion is simply its mass (in mass units, Daltons or 'Da') divide by its charge (in charge units). Do not use SI units of mass and charge to calculate this ratio! The mass-to-charge ratio is usually on the x-axis of a mass spectrum and is usually indicated by either 'm/z', 'm/e' or 'Da/e'.

Example: A molecule of phenol has a formula C₆H₆O and therefore a mass of 94 mass units (O=16, C=12 and H=1).

If it holds a single charge by loss of one electron it will have a mass to charge ratio of 94/1 = 94.
If it holds a single charge by protonation (gaining of H⁺) it will have a mass to charge ratio of (94+1)/1 = 95.

It is also possible to have multiply charged ions in your mass spectrum, especially with electrospray ionisation and MALDI (see later). In these cases your mass-to-charge ratio will be lower than the mass because the charge is greater than 1.

Example: A molecule of the protein myoglobin has a mass of 16,950 Da. When ionised by electrospray ionisation it will gain several cations and the distribution of the number of cations is roughly gaussian.

If it holds 15 charges (by protonation) it will have a mass to charge ratio of (16950+15)/15 = 1131.
If it holds 16 charges (by protonation) it will have a mass to charge ratio of (16950+16)/16 = 1060.
If it holds 16 charges (by 15 protons and 1 sodium) it will an m/z of (16950+15+23)/16 = 1061.8

6. What are meant by the terms 'mass precision', 'mass measurement accuracy' and 'mass resolution'?

Mass precision is a measure of how good the instrument you are using is at measuring a mass. This is physical limit of the design of the instrument and/or experiment you are performing and cannot be corrected for by altering (improving) the calibration of the instrument. This is a fixed experimental error and it is good science to quote it with mass data (although most people don't do this).

For example: Consider a particular instrument set up in a particular way which might have a quoted mass precision of +/- 0.0001 Da. This would mean that if you measured the parent ion of phenol as 94.0419 Da this would actually have a measured mass of 94.0419 +/- 0.0001 Da. A better way to express this error or uncertainty is 94.0419(1) Da.

Mass measurement accuracy is the observed difference between the experimentally measured mass and the theoretical mass of a particular ion. To obtain a good statistical error, it is good science to make measurement repetitions (5 times or more is good), although it is more usual nowadays to measure 20 or 30 quick repetitions and average these.

For example: If you where measuring the mass of phenol and obtained the following 5 mass measurement repetitions: 94.0419, 94.0417, 94.0418, 94.0417, 94.0416, then you could quote the mass of phenol as 94.04174 +/- 0.00022 Da (where 0.00022 is the standard deviation from the mean value). Remember this is the experimental error and doesn't included the fixed instrumental error above. It might be possible to improve the mass measurement accuracy (or error) by altering the calibration of the instrument.

Mass resolution is simply the ability of the instrument to separate ions of similar mass. It has nothing to do with mass precision or mass accuracy - although these terms are often confused. Resolution is often related to the mass range you are scanning over or to the signal intensity of the ions. If your resolution is too low to separate two ions of similar mass, then in some cases shortening the mass range and reducing the ion intensity (by closing slits or by diluting samples) might increase the resolution. The maximum mass resolution is a fixed specification of the instrument being used.

7. What is the difference between 'high resolution' and 'low resolution'?

Low resolution usually applies to a mass spectrum with unit mass resolution - i.e. neighbouring mass values are fully resolved form each other (masses are usually quoted as nominal masses no matter how accurate they are). This would mean that ions of the same nominal mass would be coincident in the spectrum and therefore inseparable.

High resolution usually applies to a mass spectrum where coincident ions are fully resolved. In practice for standard accurate mass determinations, a resolution of about 5000 (50% valley) - i.e. neighbouring peaks are fully resolved at 50% of their height. This would result in peak widths of 0.2 at 1000 m/z and 0.1 at 500 m/z. This is usually good enough to produce masses with a precision of +/- 5 ppm - assuming that the calibration is accurate.

Ultra-high resolution is becoming more common with increased availability of FT-ICR instruments. In these cases it is routine to measure masses at a resolution of 30,000 to 50,000. This would enable you to fully resolve ions to 0.01 at 300 to 500 m/z.

8. What is the difference between 'average mass', 'nominal mass', 'exact mass' and 'accurate-mass'?

The average mass of an atom or molecule is just the weighted mean of the isotopes that make it up. This is the mass value used for synthetic chemistry and is usually irrelevant in mass spectrometry. For example the average mass of phenol is 94.1112 Da. However, if you are using a low resolution technique to measure a large analyte, then you would probably see a broad peak where the peak top is a measure of the average mass. This is especially true in protein mass spectrometry.

The nominal mass of an analyte is the rounded total of all the most abundant isotopes that make it up. For example the nominal mass of phenol of 94 Da.

The exact mass of an analyte is the total of the all the most abundant isotopes that make it up. For example the accurate mass of phenol is 94.04186 Da. This is also called the theoretical mass of an analyte.

The accurate mass of an analyte is the experimentally determined mass performed in a way to determine or confirm the molecular formula. In the example in point 6 above, the accurate mass of phenol was measured as 94.04174 +/- 0.00022 Da. This would give a mass error 1.3 +/- 1.1 ppm (parts per million). This would be considered a good accurate mass measurement. This though ignores the precision of the instrument. If the mass measurement precision was 0.0001 Da (see above) then this would change the mass accuracy to 1.3 +/- 2.1 ppm.

9. What are 'isobaric' ions?

Isobaric ions are simply ions that have the same nominal mass but different exact mass. For example N₂, C₂H₄ and CO all have a nominal mass of 28 Da. Their exact masses are: N₂ = 28.00615 Da, C₂H₄ = 28.0313 Da and CO = 27.99491. A resolution of about 2500 is required to fully resolve these three ions.

10. What are isotopes and how do they affect a mass spectrum?

Isotopes are atoms of an element with a different number of neutrons. These play a very important role in mass spectrometry. For example, C-13 occurs at about 1% of the natural abundance of Carbon. This would mean that for every 100 carbons in a molecule, there would statistically be one C-13. Therefore the relative height of the +1 m/z peak for a molecular ion in a mass spectrum is a reasonably accurate measure of the number of carbons in the molecule. This assumes there are no metals present - as many metals have a complex distribution of isotopes (e.g. Tin or Ruthenium). Famous isotopic ratios are Chlorine, Bromine and Silver. When an analyte contains a complex distribution of isotopes, the observed isotope distribution will be unique and by comparison with the theoretical isotope distribution (calculated at the same resolution) is a very useful method of double checking the formula of the ion being measured.