Mass Spectrometry Notes

Mass Spectrometry: Basic Concepts and Application

Aim and Learning Outcomes

• Aim: To provide an introduction to the theory and practical aspects of mass spectrometry and its application to inorganic and organic compounds.
• Learning Outcomes: At the end of this course, students should be able to:
  • Interpret simple mass spectra and use the data to suggest possible structures of organic and inorganic compounds.
  • Predict (quantitatively) the spectra of specified compounds.
  • Describe various ionization techniques and mass analyzers.
  • Describe how the resolution of a mass spectrometer is assessed.

Key Questions Addressed

• Why mass spectra can be obtained.
• How mass spectra are obtained.
• What information mass spectrometry gives us.
• How to use that information for characterization of inorganic and organic compounds.

Recommended Texts

• Inorganic Chemistry:
  • C. E. Housecroft & A. G. Sharpe, Inorganic Chemistry, 4th edition, Pearson, 2012.
  • Chapter 4, pages 93 – 98
• Organic Chemistry:
  • W. H. Brown, B. L. Iverson, E. Anslyn & C. S. Foote, Organic Chemistry, 7th edition, Cengage Learning, 2013.
  • Chapter 14, pages 557 – 578
  • Electronic copies of individual chapters may be purchased for $5.95.

More Advanced Reading

• W. Henderson & S. McIndoe, Mass Spectrometry of Inorganic and Organometallic Compounds, Wiley, 2005
• Analytical Chemistry: A Modern Approach to Analytical Science, eds. R. Kellner & H. M. Widmer, Wiley, 2004

Lecture Outline

• Lecture 1: Introduction to Mass Spectrometry
• Lecture 2: Ionization Techniques
• Lecture 3: Mass Analysis
• Lecture 4: Isotopes and Isotope Patterns
• Lecture 5: Mass Defect and Accurate Masses

Lecture 1: Introduction

• An atom's mass depends on the number of sub-atomic particles it has.

• Approximate atom:

  • Electron (charge -1, mass $9.1093897 \times 10^{-28}$ g)
  • Nucleus:
    • Proton (charge +1, mass $1.6726231 \times 10^{-24}$ g)
    • Neutron (charge 0, mass $1.6749286 \times 10^{-24}$ g)

• The mass of atoms and molecules may be expressed in Daltons (Da) or atomic mass units (u).

  • $1 \text{ Da} = 1.6605389 \times 10^{-24}$ g
  • The mass of the neutron is approximately 1 Da.
  • The mass of the proton is approximately 1 Da.
  • The mass of the electron is approximately 0 Da.

• The Dalton value for an atom or molecule is the same as the relative atomic mass (atomic weight) or relative molecular mass (molecular weight).

  • RAM of carbon is 12, and its mass is 12 Da.
  • RMM of decane is 142, and its mass is 142 Da.

• Knowing the mass of an unidentified molecule helps in identifying it.

  • If an alkane has a mass of 142 Da, it is decane ($C<em>{10}H</em>{22}$), but not necessarily a specific isomer.

• Atoms and molecules have very small masses (approximately $1 \times 10^{-21}$ g).

• This mass is too small to weigh using a typical balance. Even a quartz crystal microbalance can only weigh down to $1 \times 10^{-9}$ g.

• Mass Spectrometry:

  • A technique for measuring the mass of atoms and molecules.

• Ions can be moved by an electric field:

  • Force is dependent on the charge times the electric potential of the applied field: $F = z "." V = m "." a$
  • Acceleration is inversely proportional to the mass to charge ratio: $a = V (z/m)$

• Ions can be moved by a magnetic field:

  • Force is dependent on the charge times the strength of the applied field times the velocity of the ions: $F = z "." B "." v = m "." a$
  • Acceleration is inversely proportional to the mass to charge ratio: $a = B "." v (z/m)$

• Ions can be deflected by magnetic and electric fields.

• Ions can be separated by mass-to-charge ratio, m/z.

• If ions are singly charged ($M^+$ or $M^-$), their position after deflection is a function of their mass.

• Ions are easy to detect because a stream of them will generate a current.

• A mass spectrometer:

  • Generates gas-phase ions.
  • Separates them according to mass-to-charge (m/z) ratio.
  • Counts the number of ions of each m/z.

• Three main methods of sample introduction:

  • Batch inlet (for volatile samples).
  • Direct inlet (for involatile samples).
  • Gas chromatograph.

• Batch Inlet:

  • Ideal for volatile samples (gases or volatile liquids).

• Direct Inlet:

  • Ideal for involatile samples.

• Gas Chromatograph:

  • Interfaces to ionization chamber.

• Established:

  • The mass of an atom cannot be measured directly.
  • Streams of ions can be deflected by electric fields.
  • Streams of ions can be deflected by magnetic fields.
  • The deflection is dependent on the mass-to-charge ratio.
  • Ions can be detected relatively easily.
  • Samples can be introduced into the spectrometer by various methods depending on the form of the sample.

Lecture 2: Ionization Techniques

• How can ions be generated in the gas phase?

  • Donation of an electron --> M-
  • Knocking off an electron --> M+
  • Loss of a cation (e.g., H+) --> M-
  • Loss of an anion (e.g., Cl-) --> M+
  • Addition of a cation (e.g., Na+) --> M+
  • Addition of an anion (e.g., HCO2-) --> M-

• Molecules and ions are in the gas phase at low pressure. Chemical reactions may differ from those in solution (high pressure).

• Molecules and ions can receive greater energy than normally expected (e.g., X—Y ca. 100 to 600 kJmol-1).

• Four methods of ionizing the gaseous sample:

  • Electron impact (EI)
  • Chemical ionization (CI)
  • Fast atom bombardment (FAB) and liquid secondary ion (LSIMS)
  • Electrospray (ESI)

• Electron Impact (EI):

  • Widely used in organic mass spectrometry.
  • Works best on small, low molecular mass (up to 1500 Da), volatile, thermally robust analytes.
  • Gas-phase sample introduction is ideal for coupling to gas chromatograph (GCMS).
  • Essential for low molecular mass non-polar analytes, e.g. hydrocarbons, where no other ionization technique will ionize the sample.

• Mechanism of EI:

  • Heated metal filament (Re or W) emits electrons accelerated toward a positively-charged target.
  • Electrons strike vaporized sample molecules.
  • Energy of the electrons (70 eV) is much greater than the ionization energy of the molecule (typically 10 eV).
    • $1 \text{ eV} = 1.602 \times 10^{-22} \text{ kJ} \equiv 96 \text{ kJmol}^{-1}$
  • This produces a molecular radical cation, [M]+•, which is left with excess energy, which can fragment by a variety of pathways.

• Fragmentation patterns provide a ‘fingerprint’ for organic compounds, cross-referenced against an electronic library for substance identification.

• To obtain molecular weight information, use a soft ionization technique.

• Strengths:

  • Well-established, reproducible mass spectra.
  • Useful for virtually all volatile compounds.
  • Fragmentation patterns provide useful structural information (‘fingerprints’).
  • Can be coupled to a gas chromatograph.

• Weaknesses:

  • Produces only cations.
  • Sample must have some volatility.
  • Molecular ion may be weak or unobservable.
  • Low mass range (< 1500 Da).

• Chemical Ionization (CI):

  • The source is filled with reagent gas and subjected to a high energy electron beam. The gas forms ions which react with gaseous analyte molecules to form analyte ions, usually through proton transfer.
  • At high vacuum ($10^{-6}$ mbar), molecules and electrons form ions as in an EI source, e.g. $CH<em>4 + e^- \rightarrow CH</em>4^{"+} + 2e^-$.
  • At higher pressure ($10^{-3}$ mbar) the initially formed ions react with neutral molecules to form secondary ions: $CH<em>4^{"+} + CH</em>4 \rightarrow CH<em>5^+ + CH</em>3^{"}$.
  • With a substrate M, proton transfer can give a pseudomolecular ion: $M + CH<em>5^+ \rightarrow [MH]^+ + CH</em>4$

• Produces less fragmentation than EI, and the pseudomolecular ion, [MH]+, is often more abundant

• [MH]+ is not a radical ion, so radical-promoted fragmentation pathways are suppressed.

• Strengths:

  • May give molecular weight information via pseudomolecular ion [MH]+.
  • Less fragmentation than EI.
  • Can be performed at atmospheric pressure (APCI).
  • APCI can be coupled to high performance liquid chromatograph (HPLC).

• Weaknesses:

  • Sample must have some volatility for CI (not required for APCI).
  • Fragment ions insufficiently reproducible for library searching.
  • CI - low mass range (< 1000 Da).

• Fast Atom Bombardment (FAB) / Liquid Secondary Ion Mass Spectrometry (LSIMS):

  • A fast-moving beam of atoms/ions blasts the matrix and analyte into the gas phase.
  • The secondary ions that are mass analyzed are either originally charged or acquire a positive charge from protonation (or association with another charged species such as Na+) or a negative charge by deprotonation.

• Strengths:

  • Fast and simple.
  • Useful for a wide range of samples.

• Weaknesses:

  • Relatively soft ionization, but superseded by even softer ESI.
  • High chemical background noise, especially at low m/z (< 300).
  • Analyte must be soluble and stable in matrix.
  • Hard to handle air-sensitive samples.

• Matrix Assisted Laser Desorption Ionization (MALDI):

• Requirements for the matrix:

  • Low volatility
  • Absorb light at the laser wavelength
  • Promote analyte ionization
  • Soluble in a common solvent as analyte
  • Co-crystallize with sample
  • Solid

• Strengths:

  • Rapid and Convenient.
  • Relatively soft ionization technique.
  • High mass range.

• Weaknesses:

  • Relatively soft ionization, but superseded by even softer ESI.
  • Not compatible with liquid chromatography (LC).
  • Singly-charged ions irrespective of sample.

• Electrospray Ionization (ESI):

• Strengths:

  • Rapid and Convenient.
  • Extremely soft ionisation technique.
  • Suitable for charged, polar or basic compounds.
  • Multiply-charged ions often seen.
  • Compatible with LC.
  • Extremely popular.

• Weaknesses:

  • Unsuitable for non-polar compounds.
  • Sensitive to contamination.

• Established:

  • There are various ways to generate ions, both cations and anions, in the gas phase.
  • Electron impact is a ‘hard’ method.
  • Electrospray is the ‘softest’ method.

Lecture 3: Mass Analysis

• Established:

  • There are various ways to generate ions, both cations and anions, in the gas phase.
  • Streams of ions can be deflected by electric fields.
  • Streams of ions can be deflected by magnetic fields.
  • The deflection is dependent on the mass-to-charge ratio.
  • Ions can be detected relatively easily.

• Mass analyzers separate ions according to their m/z ratios.

• All methods employ electric fields, sometimes in conjunction with magnetic fields.

• Five main types of mass analyzers:

  1. Sector
  2. Quadrupole
  3. Ion trap
  4. Time-of-Flight (TOF)
  5. Fourier Transform Ion Cyclotron Resonance

• Sector

  • Ions leaving the source are accelerated into a magnetic sector, and a magnetic field is applied perpendicular to the ion beam.

• Strengths:

  • High resolution and sensitivity.
  • Good mass range (up to m/z 4000).

• Weaknesses:

  • Not well suited for pulsed ionization techniques e.g. MALDI.
  • Coupling with atmospheric pressure sources (ESI, APCI) is complicated.
  • Large and expensive.

• Quadrupole

  • Ions are separated as they pass along the central axis of four parallel rods.

• Strengths:

  • Compact, easy to clean.
  • Inexpensive.
  • Fast scanning.
  • Good reproducibility.
  • Easy coupling to atmospheric pressure sources (ESI).
  • Reasonable mass range (up to m/z 4000).
  • Very popular.

• Weaknesses:

  • Limited resolution.
  • Not well suited for pulsed ionization (MALDI).

• Time-of-Flight (TOF)

  • Measurement of the times taken for ions of different m/z to travel the length of an evacuated tube.
  • Recent improvements in digital timing electronics and instrument design have made high-resolution TOF analyzers quite affordable.

• Strengths:

  • Simple.
  • Unlimited mass range.
  • Rapid mass analysis.
  • Compatibility with MALDI.
  • High resolution.

• Weaknesses:

  • High vacuum required.
  • Pulsed ion source required.

• Lecture Summary:

  • Spatial separation of ions using electric and magnetic fields
  • Temporal separation of ions after acceleration
  • Sector mass analysis
  • Quadrupole mass analysis
  • Time-of-flight mass analysis

Lecture 4: Isotopes and Isotope Patterns

• Isotopes

  • Atoms of the same element which possess different numbers of neutrons.
    • e.g. Hydrogen – one proton and one electron
      • $^1H$: no neutrons
      • $^2H$: one neutron (Deuterium)
      • $^3H$: two neutrons (Tritium)
    • Uranium – 92 protons and 92 electrons
      • $^{235}U$: 143 neutrons
      • $^{238}U$: 146 neutrons

• Isotopologues

  • Molecules of the same elements which contain different isotopes of one or more atoms.
    • e.g. $CHCl<em>3$ and $CDCl</em>3$

• Isotope Pattern

  • The natural abundances of isotopes give rise to patterns for most elements:

• The number of atoms of an element in a compound may be indicated by the pattern of the mass spectrum.

Lecture 5: Mass Defect and Accurate Masses

• Isotopic Mass

  • Mass of the proton, mp: $1.6726231 \times 10^{-24}$ g $\equiv$ 1.007276 gmol-1
  • Mass of the neutron, mn: $1.6749286 \times 10^{-24}$ g $\equiv$ 1.008665 gmol-1
  • Mass of the electron, me: $9.1093897 \times 10^{-28}$ g $\equiv$ $5.4858 \times 10^{-4}$ gmol-1

• Mass Defect

  • [Z  mp + Z  me + (A - Z)  mn] - mass of atom = mass defect

• The resolution of a mass spectrometer represents its ability to distinguish ions of different m/z, manifested in the sharpness of the peaks.

• Mass resolution

  • R = m/Dm, where m is the mass of the ion, and Dm is the distance to another peak overlapping such that there is a 10% valley between the peaks:

• It is often more convenient to define Resolution on a single peak, in which case Dm is the full width of the peak at 5% of its maximum intensity:

• The mass accuracy of a spectrometer is the difference between the calculated mass of an ion and its observed mass, expressed relative to the observed mass.