L6: Mass spectrometry

nguyen-thi-kieu.oanh@usth.edu.vn

1
 OBJECTIVES & OUTCOMES

Objectives:
To introduce basic understanding of mass spectrometry

Outcomes: At the end of this lesson, students are able:

To describe the principle of mass spectrometry
To present basic parts of a mass spectrometer
To answer the question how to interpret the mass spectrum?

2
 OUTLINES

 What is mass spectrometry?

 History
 Theory
 Mass spectrometer
 Resolution
 The MS spectrum

3
 1. What is Mass spectrometry?

Basic terms:
 Spectroscopy: study of interaction of electromagnetic radiation with matter
 Spectrometry: use of spectroscopy as a analytical tool
 Mass spectrometry: In simple terms, a mass spectrometry measures the masses
within a sample
 Mass spectrum: It is a histogram usually acquired using an instrument. A mass
spectrum is a lot of signal as a function of mass-to-charge ratio

Coupled with chromatography it is one of the most powerful

technique in Bioanalytical chemistry

4
 1. What is Mass spectrometry?

Basic principle
 A charged particle moving in a magnetic field is deflected
Degree of deflection depends on the mass to charge ratio
 A mass spectrometer measures mass-to-charge ratio m/z
 By separating ions in vacuum
 If z=1 it measures directly the mass

See video

5
 2. MS: History

 J. J. Thompson: early 1900s

 Dempster and Aston
 Devised modern mass spectrometry
 Around 1918-1919

Ions are accelerated by the electric field V

Separated by the magnetic field B
(perpendicular)

6
 3. MS: Theory

The history of isotopes parallels that of mass spectrometry

 Average mass = molecular mass of a molecule
 The chemist used to weigh a compound – depends on the value of the isotope
natural abundance
 It may vary slightly accordingly
 However, in mass spectrometry
 We do not see the average mass
 We see the mass of the isotopes

7
 3. MS: Theory

 Example: bromine
 In Mass spectrometry we never see average mass 79.9
 We see the isotopes 78.9 and 80.9

8
 3. MS: Theory

 Monoisotopic mass
 A molecule is made of: 12C, 1H, 14N, 16O,...
 The mass obtained with the most abundant isotopes is called the monoisotopic mass
 Reserpine: Monoisotopic mass = 608.273376
 Nominal mass
 Is obtained by the chemical composition: using integers for the atom, C=12, O=16, N=14
 Reserpine: nominal mass = 608

“If you understand the different mass definitions

you understand half of mass spectrometry”

9
3. MS: Theory

10
 3. MS: Theory
 Isotopic distribution
 Note that the lightest isotope is also the most abundant one for these elements.

Normally, the most abundant

ion have monoisotopic mass M !

11
 3. MS: Theory

 Isotopic distribution
Isotope Mass % Abundance
 Sulfur has a big impact on the isotope distribution. But it is not 34S 33.967868 4.21
always present in a biological compounds (only the amino acids 13C 13.003355 1.07
Cystein or Methionin contain sulfur). 33S 32.971459 0.75
 13C is most abundant, followed by 15N. 15N 15.000109 0.37
18O 17.999159 0.20
 The heavy isotopes 18O and 34S lead to “+2” peaks.
17O 16.999131 0.038
 Note that 17O and 2H are very rare. 2H 2.014102 0.0115

The isotopic distribution is due mainly to 13C (1.07%) calculated by the binomial
distribution: number of carbon in the molecule x 1.07%.

12
 3. MS: Theory

 Isotopic distribution
 Number of atoms in a molecule
increases the probability of the entire
molecule containing at least one
heavy isotope

 An example of glucagon: The most abundant isotopic species here is not the monoisotopic
mass, but appears two atomic mass units higher, reflecting the contribution from 13C carbon
atoms as well as contribution from 15N and 18O.
13
 4. The mass spectrometer

3 main parts + software

ionization, separation of ions, detection of ions

14
 4. The mass spectrometer

4.1 Ionization
The molecule has to be converted to an ion by ionization source.
Types of ionization:
 EI: Electron ionization (also called electrical impact)
 CI: Chemical ionization (also called chemical impact)
 FAB: Fast atom bombardment
 ESI: electrospray ionization
 MALDI: Matrix – Assisted Laser Desorption Ionization

15
 4. The mass spectrometer

4.1 Ionization
Electrical impact (EI) ionization

M + e-  M+. + 2e-
 High energy ionization
 It is one of the oldest method in which the molecule is hit in the source by a beam of
high energy electrons (20-70 eV). The source has to be in vacuum since it is a high
energy collision the molecular ion gives many fragment ions.
 This ionization mode is good for structure identification. But we may not see the
[molecular ion] because we use very high energy.
16
 4. The mass spectrometer

4.1 Ionization
Chemical impact (CI) ionization

Reagent gases: Methane, ammonia, isobutane

CH4 + e-  CH4+. + 2e-
CH4+. + CH4  CH5+ + CH3.
CH5+ + M  MH+ + CH4
 Chemical ionization is a lower energy process than electron ionization (EI)
 Less or sometimes no fragmentation, and usually a simpler spectrum  limit structural elucidation
 Very soft ionization thus useful in cases where EI cannot used to form in large enough quantities of
molecular ions.
17
 4. The mass spectrometer

4.1 Ionization
Electrospray ionization (ESI)
 Electrospray generates fine droplets by applying an electrical voltage across the sample which
comes through a fine metal needle.

 The molecule remains largely intact during the ionization process.

 It is soft ionization because it needs lower energy than EI. Therefore this source is at
atmospheric pressure and we often see the molecular ion in form of M(H+)n; M(H+)n(Na+)n...

18
Source: http://www.chm.bris.ac.uk/ms/esi-ionisation.xhtml
 4. The mass spectrometer

4.1 Ionization
MALDI: Matrix – Assisted Laser Desorption Ionization
 For big molecules: 300 000 Da

 The sample is mixed with a matrix on a metallic plate.

 The matrix contains UV-absorbing molecules compounds, chosen to

absorb the laser wavelength. Laser excites the matrix that goes into
the gas phase with the sample.

 It also a soft ionization and often gives M+H+.

19
 4. The mass spectrometer

4.2 Separation
After ionization, ions are accelerated and separated in a magnetic field.
There are several types:
 Quadrupole
 Time of flight TOF
 Reflector TOF
 Ion trap

20
 4. The mass spectrometer

4.2 Separation
Quadrupole: The ions fly in four cylindrical rods, set parallel to each other.
It is responsible for filtering sample ions, based on their mass-to-charge ratio (m/z).
Ions are separated based on the stability of their trajectories.

 Used in most analytical mass spectrometers

 Trajectories is complicated: Mathieu equation
 Voltage applied to the rods determines the ion

21
 4. The mass spectrometer

4.2 Separation
 Quadrupole
 Time of flight TOF
 Kinetic Energy = z.e.V ½ mv2 = z.e.V
 The transit time (t) through the drift tube is L/v where L is the length of the drift
tube.
 The ions are separated by the time they spend in the tube to reach the detector

22
 4. The mass spectrometer

4.2 Separation
- Quadrupole
- Time of flight TOF
- Reflector TOF: At the end of the drift tube an opposite electric field is added. The
principle is the same with TOF.

23
 4. The mass spectrometer

4.2 Separation
- Quadrupole
- Time of flight TOF
- Reflector TOF
- Ion trap: The ions are orbiting in a trap. You can select the one(s) you want by giving a
stable resonant trajectory in the ion trap.

24
 4. The mass spectrometer

We can combine these techniques

 Tandem mass spectrometry: MS/MS, MS/MS/MS
 Hybrid instruments: Triple quadrupole, Q TOF, TOF TOF, Q – IT, Q – Orbitrap...

25
 4. The mass spectrometer

4.3 Detection
- The final element of the mass spectrometer is the detector.
- The detector records either the charge induced or the current produced when an ion passes by
or hits a surface.
- Detection in mass spectrometry: Electron multiplier

26
 5. Resolution

 Resolution measures of the ability to distinguish two peaks of slightly different mass-to-
charge ratios ΔM, in a mass spectrum.
 There are several ways to calculate resolution:
– Full width at half maximum (FWHM): used in TOF: m/∆m
– 10% and 50% valley: m1/∆m

 The resolution depends on the mass, so the resolution

should always give the corresponding mass.

27
 5. Resolution

 High resolution instruments:

- LTQ Orbitrap: Hybrid Mass Spectrometer: R > 100 000
- ESI-Qq-oaTOF: R > 40 000.
 Application of high resolution
 Determination of isobaric compounds
o Clonazepam: monoisotopic mass 315.041077
o Bromazepam: monoisotopic mass 315.000702

28
 6. The mass spectrum

The output of mass spectrometry is a mass spectrum which is usually simplified into a
"stick diagram". This shows the relative current produced by ions of varying mass/charge
ratio.

The mass spectrum gives information about:

 the structure,
 the molecular ion (confirms the MW) and
 the fragments (depending on the structure).
29
 6. The mass spectrum

Determination of the molecular mass:

 In soft ionization methods: CI, Electrospray and MALDI the molecular mass is assessed by the
presence of adduct ions:
 Hydrogen: {M+H}+
 Alkali metal: {M+Na}+, {M+K}+,
...
 In EI, ion trap: molecular ion is M+

30
 6. The mass spectrum

Fragmentations
 Depend on the structure
 For example: C2H6O
 Based on fragments in the mass spectrum, we can predict the structure of molecule.

15

29

19
48

31
 6. The mass spectrum

Fragmentations
 Depend on the structure
 For example: Pentane & 2-methylbutane

32
 6. The mass spectrum

Isotope patterns
 Isotope patterns with Cl and Br: Mass spectrometers are capable of separating and detecting
individual ions even those that only differ by a single atomic mass unit.
 79Br: 81Br,intensity 1:1 and 35Cl: 37Cl, intensity 3:1 where peaks at "M" and "M+2" are obtained. The
intensity ratios in the isotope patterns are due to the natural abundance of the isotopes.

33
 Take-home messages
 Mass spectrometry measures ions (m/z) in the gas phase
 The monoisotopic mass of a molecule is the lowest mass formed by 12C, 1H, 14N, 16O... Mass
spectrometry does not measure the average mass which is a mixture of the monoisotopic mass and
masses from isotopes (mainly 13C).
 The mass spectrometer has 3 main parts:
 The ion source
 The ion separator
 The ion detector
 Electrospray ionization and Matrix assisted laser ionizations are soft ionizations and give the MH+ ion
with little fragmentation.
 More energetic ionization (ESI) gives extensive fragmentation which can be used for structure
elucidation.
34
Thank you for your attention!

35