Peter J Baugh

1
Analytical Mass Spectrometry

• Peter J Baugh
• Quay Pharma Training Progamme
• Day 4, September 28, 2004

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Analytical Mass Spectrometry

• Mass spectrometry is an extremely powerful
  analytical instrumental tool in its own right but
  also, and importantly, when coupled to a range of
  introductory and/or chromatographic systems, such
  as GC (SFC) and LC. It is used by chemists,
  biochemists, biologists and physicists in many
  fields analytical/environmental, molecular
  biological, toxicological, clinical, medical and
  physical
• MS has been at the forefront of the recent high
  powered research to elucidate/characterise the
  Human Genome and without its capability in
  analysis no advances would have been made in this
  field.
• This training session is designed to introduce MS
  as a stand alone analytical tool and when
  interfaced to chromatographic systems. The
  following areas will be covered
• Theory of MS ion production, processes and
  mechanisms
• Instrumental configurations ionisers,
  analysers, detectors, data systems, scan modes
  and functions
• Introductory and interfaced systems
• Applications in selected areas

3
Introduction to MS

• Background Aston in 1920s designed a mass
  spectrograph using a magnet to mass analyse and
  photographic plate to detect positive ions
  produced by ionisation of gases and vapours.
  Using this mass spectrograph he identified in
  excess of 200 isotopes of a wide range of
  chemical elements and received the Nobel prize
  for this work. Today we use his foundation work
  as a basis for the field of mass spectrometry (a
  term now used more frequently rather than the
  older term of mass spectroscopy the latter
  inferring that a spectroscopic detector is
  utilised).
• Nowadays, the analysers are more wide ranging in
  type and in combination as follows
• Magnetic (B) and electrostatic (E) analysers in
  combination, EB or reverse geometry BE
• Quadrupole (Q) and ion trap (IT) electric
  fields only
• Time of flight (TOF)
• Ion cyclotron resonance (Fourier transform MS)
• In addition both hybrid and tandem analyser
  systems have been designed to conduct analysis
  using more complex scan modes, EBqQ, QTOF,
  QqQ, EBBE

4

• Theory of Mass Spectrometry
• Much work has been carried out on the theory of
  ion production and ion processes. Until the
  1970s only positive ion mass analysis could be
  conducted because the ionisation modes , such as,
  EI, FD (FI) produced only positive ions in
  sufficient abundance from solids bombarded by
  electrons emitted from a hot filament, or by
  desorption - electric field application. Sample
  introduction was via a solids probe. Strictly
  speaking electrons generated could be captured by
  molecules in the ionisation source but with
  almost zero efficiency because of the large mean
  free path high vacuum, very low pressure
  atmosphere. In fact, the efficiency of
  ionisation to generate positive ions is only a
  few .
• A positive ion, M., are generated from a
  molecule of a chemical solid, M, in Electron
  Impact ionisation with highest efficiency by
  employing electrons with Ee 70 eV as follows
• M e- ? M. 2e-
• If the radical cation formed is unstable which is
  often the case because it has too much kinetic
  energy imparted to it during the ionisation
  process it can break up in a number ways to
  produce secondary ions and neutral radicals or
  ions and molecular products.
• M. ? A B. or A. B or C H2
• The secondary ions, A , B, or C, are
  accelerated from the ion source using an applied
  voltage Vacc (high or low depending on the mode
  of MS) , mass analysed and detected. Some ions
  can also break down in flight metastables, and
  such ions can be analysed also by linked scanning
  (EB or BE instruments only).

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• Theory of Mass Spectrometry
• Stabilisation and fragmentation of positive ions
  produced by EI
• Mechanisms of positive ion breakdown are numerous
  and vary depending on the stability of the ion
  and the nature of the molecule. The molecular
  ion produced by EI is commonly unstable and the
  breakdown or fragmentation leads to more stable
  ions which vary in abundance and the distinctive
  pattern characteristic of the molecule is termed
  the mass spectrum (relative abundance versus m/z
  ratio). The most stable ion is termed the base
  ion or the base peak in the mass spectrum (100
  relative abundance). The structure of a molecule
  can often be deduced from the mass spectral
  fragmentation pattern and molecules with similar
  structures show similar mass spectra, however,
  knowledge of the mass (m/z) of the molecular ion
  is required for confirmation. An homologous
  series of compounds exhibit similar mass spectra
  and the same base ion (100).
• For example
• N-alkanes. m/z, 57, C4H9 , the butyl ion
  N-alkenes, m/z , 55, C4H7, butylene ion
• fatty acid methyl esters, m/z, 74, a
  stabilised ion via a McClafferty rearrangement,
• CH3O C CH2
• OH
• Aromatic compounds exhibit molecular ions or
  stabilised fragment ions with the aromatic
  structure intact
• Other ionisation modes
• FD generates, preferentially molecular ions or
  stabilised ions by elimination of water.
• Pseudo molecular positive and negative ions and
  adduct ions are form by other ionisation
  processes CI, FAB, TSP and ESI, APCI, MALDI
  (Laser desorption/ionisation), PD (plasma
  desorption 252Californium)

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• Mass Spectrometric instrumentation
• Essentially, a mass spectrometer comprises an ion
  source, mass analyser(s) and a detector in a
  high vacuum system, almost always nowadays
  electronically-interfaced to a data system (DS).
• Ion source (see schematic diagram 1)
• Sample introduction for EI in its simplest form
  is via a solids probe heated to several hundred
  0C. This type of introduction is limited to
  organic solids of up to about 600 u RMM.
  Oligomers, Peptides and proteins of high RMM
  undergo excessive fragmentation to yield low RMM
  fragment ions. Excessive decomposition occurs in
  organic solids that decompose on heating, i.e.,
  have no m.pt. The ions are generated in the ion
  source by bombardment with electrons emitted from
  a heated filament e.g., rhenium wire, to which a
  voltage is applied - 150 V. The electron energy,
  Ee, can be varied (10 120 eV) and the molecular
  ion stability is inversely proportional to Ee .
  In an EI source the electrons are collected (go
  to earth). A high accelerating voltage is
  employed for EI in EB or BE MS and a low voltage
  is used for quadrupole MS. In the CI source the
  electrons are not collected.
• Mass analysers
• Magnetic sector/electric sector analysers MS is
  fundamentally based on magnetic sector analysers,
  such instruments are single focussing (SF) with
  respect to mass through use of a magnet only.
  Double focusing (DF) sector instruments either
  focus ions first through the electric sector
  (ESA), energy focusing and then the magnet (B),
  mass or momentum focussing, or vice versa in
  reversed geometry, i.e., EB or BE. The DF
  systems are capable of high mass resolution
  (0.001 u or 1 ppm), scanning metastables and
  linked scan functions (based on E, B and possibly
  Vacc together).

7
Diagram 1. EI Ion Source
8

• Mass analysers continued
• Although the equation describing the relationship
  between m/z (m/e), B2, Vacc , r, the field radius
  is based on the SF magnetic sector instrument
  that for the DF sector MS is more appropriate
  which describes the relationship between m/z, B2,
  the electric sector voltage, E, the distance
  between the ESA plates, d, the field radius, r ,
  and the radius of curvature of the ESA, R.
• m/z B2 r2 d/ ER .
• It can be seen that m/z is independent of Vacc .
  Scanning is conducted with respect to E, B (or V)
  or linked E/B or B2/E , etc. The accelerating
  voltage is 2 8 kV. The magnetic field flux is
  normally scanned, exponentially downwards, where
  m mo. exp(-2bt) with scan rates being described
  in terms of seconds per decade of mass. Until
  the 1980s the magnet scanning was hampered by
  long reset times imposed by hysteresis of the
  magnet the magnetic cycle involves scan-up,
  scan down and reset phases. Thus the scanning of
  a quadrupole was much faster at that stage in
  time. The introduction of low hysteresis magnets
  (laminated) with rapid reset times allowed
  improved scan speeds (lt 0.1 sec per decade)
  compatible with quadrupole systems.
• See schematic diagram -2 and 3 of DF magnetic
  sector instrument

9
Diagram 2B ESA of a Double Focusing MS
Diagram 2C Normal Geometry DF MS energy and
momentum focusing to increase mass resolution
10
Diagram 3 Normal Geometry DF MS showing ion
optics
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• Mass analysers continued
• Quadrupole mass analyser The basic quadrupole
  mass analyser is an electric sector device
  depending only on generation of electric fields.
  The analyser comprises circular (hyperbolic) four
  rods making up the quadrupole in which
  diametrically opposed rods (x and y directions)
  are electrically coupled to a combination of dc
  and rf amplitude voltages, U and V (see schematic
  diagram - 4).
• To operate the analyser to enable mass analysis a
  combination of U and V must be applied to the x
  and y rods such that the ratio of U/V is
  constant. Varying the voltages over a set range
  allows ions of a selected m/z to undergo stable
  z-direction oscillations in the electric field
  generated at the appropriate U/V (the quadrupole
  is termed a mass filter for this reason). The
  Mathieu equations describing the trajectory in
  the combined field in x and y directions are not
  discussed here. However, it is useful to note
  that the application of U and V voltages is
  governed as follows
• x-axis rods U Vcos(wt) y-axis rods -U
  Vcos(wt)
• The coswt relationship implies that the rf
  amplitude voltage,V, is 180o out of phase with
  the dc voltage.The stability diagram (triangle)
  describing the region in which ions of a selected
  m/z is stable is obtained from solutions of the
  Mathieu equations (terms a and q) and provides
  information about the conditions under which mass
  analysis can be conducted. Essentially, only
  ions of a selected m/z can be analysed at a
  particular U/V and mass selection is maximised
  when ions of different m/z attain the same
  velocity (imposed by varying the accelerating
  voltage up to 20 V applied to the ions exiting
  from the ion source, coincidently with U/V) (see
  schematic diagrams 5 and 6).
• Transmission of ions is lost towards the apex of
  the triangle where the resolution (mass
  selection) is greatest and a quadrupole mass
  analyser is essentially capable only of low mass
  resolution (unit mass accuracy) where as a DF
  magnetic/electrostatic sector analyser system is
  capable of low and high mass resolution.)

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Diagram 4 Quadrupole MS showing a. X () and Y
(-) rods coupled to dc and rf voltages forming
the mass analyser and b. ion trajectory
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• Additional Notes
• Parameters, a and q, for the Stability Diagram
• Quadrupole and Ion Trap Analysers
• The primary concern in the utilisation of a
  quadrupole device are the criteria that govern
  the stability of an ion trajectory within the
  field, i.e., the experimental conditions that
  determine whether the ion remains within the
  device or is lost to the environment. The
  trajectory of a charged particle under the
  influence of a two dimensional field is described
  as an oscillating system in which the restoring
  force is periodic in time. The system can be
  described by two parameters, a and q,
  formularised in terms of dc (U), rf (Vamp), ion
  mass (m), radiofrequency (?), charge (e) and the
  distance between the rods (ro)
• a 8neU/m (ro ?)2 and q 4neV/m (ro ?)2
• Being periodic in time, a and q could have a
  range of values but only discrete values allow
  the amplitude of the ion oscillation to remain
  bounded. Hence, it is customary to construct a
  stability diagram that reveals the range of a/q
  values for which the amplitude remains finite
  (stable) and other values for which oscillations
  become infinite (unstable).
• The a/q diagram show regions of stability that
  may be satisfied by many combinations of U, V and
  m with the assumption that ro and ? are constant.
  The mass scan lines consist of ion values having
  a constant a/q ratio. Transmission of an ion
  occurs when U and V are such that the ion has
  particular values of a and q lying within the
  stable region. A diagram in (U,V) space,
  however, is specific to particular mass to charge
  ratio, and each ion will possess a different U,V
  diagram. Thus, the mass scan will successively
  pass through the apices of a family of diagrams
  (see stability diagrams 5 and 6).

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Diagram 5 Stability Curve for ions, m1 , m2
and m3 only m2 within the region of stability,
is transmitted and detected.
Diagram 6 Stability curves for ions m1 , m2
and m3
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• Mass analysers continued
• Quadrupole analysers are said to discriminate
  across the mass range as m/z increases, i.e., the
  transmission is reduced whereas magnetic
  analysers do not exhibit this effect.
• The triple quadrupole is capable of MS-MS, see
  later.
• Mass scanning is conducted linearly upwards with
  respect to the voltage applied.
• Ion trap analyser The IT analyser can be
  described as a 3D quadrupole with three
  hyperbolic electrodes comprising a ring and two
  end caps that form the core of the instrument.
  Similarly to the quadrupole only an electric
  field is generated in the ion trap and ions
  generated are held in the ion trap when the dc
  and rf amplitude voltage are applied to the ring
  electrode. Ion ejection is initiated by selective
  ramping of the fundamental rf amplitude voltage
  that sequentially makes ion trajectories unstable
  allowing mass analysis and a mass spectrum to be
  generated by sequential ejection of fragment ions
  from low to high m/z.. Ions are ejected through
  holes in the endcap electrode and detected by an
  electron multiplier. (see schematic diagram - 7)
• There are two scan modes of operation
• mass selective stability whereby dc and rf
  voltage applied to the ring electrodes are ramped
  at a constant rf/dc ratio to allow stability and
  storage of ions of a single m/z for mass analysis
• mass selective instability whereby all ions
  created over a given time period are trapped and
  then sequentially ejected from the IT to the
  detector (EM). Thus ions of differing m/z are
  stored while mass analysis is performed. Unlike
  the mode above this mode coupled with the
  presence of helium gas (1 mtorr) in the trapping
  volume greatly improves mass resolution by
  contracting the ion trajectories to the centre of
  the trap and reducing the kinetic energy of the
  ions allowing ions of a given m/z to form a
  packet. These advances allowed the successful
  development of the commercial ITMS.

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• Ion Trapping Ions created by EI, ESI or MALDI
  are focused using an electrostatic lensing system
  into the ion trap. An electrostatic ion gate
  pulses open (-V) and closed (V) to inject ions
  into the ion trap. The pulsing of the gate
  differentiates ion traps from beam instruments
  where ions continually enter the mass analyser.
  The time during which the ions are allowed into
  the trap, termed the ionisation period, is set to
  maximise the signal while minimising the
  space-charge effects. Space-charge results from
  too many ions in the trap that cause a distortion
  of the electrical fields leading to an overall
  reduction in performance. The ion trap is filled
  typically with helium (1 mtorr). Collisions with
  helium molecules dampens the kinetic energy of
  the ions and serve to quickly contract ion
  trajectories toward the centre of the trap,
  enabling trapping of injected ions. Trapped ions
  are further focused toward the centre of the trap
  the use of an oscillating potential, referred to
  as the fundamental (rf), applied to the ring
  electrode. An ion will be stably trapped
  depending on the values of m/z of the ion, the
  size of the ion trap (r), the oscillating
  frequency (?), and Vamp . The dependence of ion
  motion on these parameters is described by the
  dimensionless parameter qz,
• qz 4eV/mr2w2
• (see schematic diagram 7 diagram - 8 ).

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Ring Electrode rf ? Vamp
End Cap Electrode tickle (ac) voltage
Diagram 7 Ion trap schematic
Diagram 8 Stability Diagram (SD) for ions in an
ion trap showing the region of overlap of axial
and radial stability. Depending on the Vamp an
ion of given m/z will have a qz value that falls
within the boundaries of the SD and the ion will
be trapped, If the qz at Vamp falls outside the
boundaries SD, the ion will hit the electrodes
and be lost.
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• Ion trap Glossary
• ac voltage- a voltage placed on the endcap
  electrodes used to induce resonance excitation
  and resonance ejection.
• bath gas, damping gas, target gas He- in the
  trapping volume at 1 mtorr serves to cool the
  kinetic energies and focus ion trajectories into
  a tight packet in the centre of the trap.
• fundamental rf- typically 1.1 MHz potential
  applied to the ring electrode, the amplitude of
  which determines the range of m/z values that can
  be trapped and is ramped to eject ions.
• high resolution- the mass scan of the instrument
  is reduced resulting in an increase in the
  density of data points per unit m/z thus
  increasing mass resolution.
• qz- dimensionless parameter that determines
  stability of ion trajectories depending upon
  their m/z ratio, the size of the trap, and the
  amplitude and frequency of the rf.
• Resonance- an ac voltage is applied to the endcap
  and the qz value of an ion of interest is changed
  until the secular frequency of the ion matches
  the frequency of the applied voltage. When
  resonance occurs, the amplitudes of the ion
  trajectories increase linearly with time. A high
  amplitude ac voltage will cause resonance
  ejection, while a low ac voltage will cause
  resonance excitation.
• secular frequency- the frequency dependent upon
  the qz value, with which an ion oscillates in the
  trap.
• space-charge effects- too many ions in the trap
  distort the electric fields, leading to
  significantly impaired performance.
• tickle voltage- an ac voltage applied to the
  endcaps during the excitiation period. The
  amplitude is generally small so as to enable
  fragmentation of the ions by collision with the
  helium damping gas rather than ejection.

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• Ion excitation and ejection Ions oscillate with
  a frequency, known as the secular frequency, that
  is determined by the values of az and qz and rf,
  ?. Resonance conditions are induced by matching
  the frequency of a supplementary potential by the
  application of a low amplitude ac resonance
  signal across the end cap electrodes causing the
  ion kinetic energies to increase and leads to ion
  dissociation due to many collisions with the
  helium damping gas. This process causes random
  fragmentation of the ion analogous to that
  obtained in TSQ MS. A mass spectrum is generated
  by sequentially ejecting fragment ions from low
  to high m/z by choosing amplitudes of the
  fundamental rf potential that sequentially make
  ion trajectories unstable. Ions are ejected
  through holes in the encap electrodes and
  detected using an electron multiplier.

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• The mass scanning is conducted in a manner
  similar to that for a quadrupole (linearly for
  m/z with respect to voltage).
• The generation of the electric field is governed
  by complex Mathieu equations that do not need to
  be discussed here.
• The nominal mass range has been extended from m/z
  650 to 70,000 greater than a quadrupole MS but
  less than a TOF-MS. Worth noting is that
  multi-stage MS-MS (or MSn where n 1 12) can
  be carried out in an ion trap directly by
  introduction of a collision gas (argon) causing
  collisionally-induced decomposition (CID) of the
  initial ions trapped. This feature is beyond the
  capability of of TSQ or TOF-MS and greatly
  increases the the structural information
  obtainable for a given molecule.
• The ITMS is extremely sensitive being able to
  detect as few as 1.5 million peptide molecules
  and this feature together with others has created
  an interest in applications of ITMS to biological
  molecules. Easy access to different modes of
  scanning for different experiments is provided
  without the need for extensive manual set-up and
  tuning and in-depth understanding of theory thus
  extending the power to biologist and biochemists.

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• Mass analysers continued
• Time of flight mass analyser The TOF analyser
  discriminates between ions of differing m/z by
  the time taken to traverse a flight tube, drift
  to the detector A pulse of ions is accelerated
  out of the source by application of a reasonably
  high voltage. Ions of all m/z have the same
  energy . Thus the velocity v decreases with
  increasing m/z and the time taken to reach the
  detector increases with m/z. The separation of
  ions of differing m/z depends on the time of
  traverse. A time signal is initiated when ions
  are pulsed and also when ions arrive at the
  detector so that the time of flight for each m/z
  can be measured.
• See schematic diagram - 9
• The velocity, v, of an ion is determined by the
  accelerating voltage. The kinetic energy is given
  by
• m2v/2 zV
• where m mass z charge on ion (electronic
  charge) and V accelerating voltage
• The time of flight of an ion, t, is given by t
  l/v where l is the length of the flight. The mass
  of ion can be calculated if the time of flight,
  the length of flight, the charge on the electron
  and the accelerating voltage are known, as
  follows
• m/z 2V2t2 / l2
• The mass range of a TOF analyser is almost
  infinite and so for this reason with MALD it has
  found application recently in the analysis of
  high mass polymers for biomolecular (protein) and
  synthetic polymer research and applications. The
  resolution of a TOF-MS depends on the time spread
  of the ion beam and was limited to ca. 2000 u.
  Recent advances employ reflectron lenses
  (electrostatic repeller field ion mirror) and
  the resulting delayed extraction allows improved
  resolution by minimising small differences in ion
  energies and resolutions of up to 12000 u
• . Higher energy ions penetrate deeper into the
  ion mirror than lower energy ions of the same m/z
  and will be turned around and arrive at the
  detector at the same time.

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Diagram 9 Reflectron TOF MS
Reflectron TOF
23

• Ionisation methods
• Ionisation of a compound to produce positive ions
  has been exemplified so far by the
  well-established conventional EI (FD) in an ion
  source under high vacuum conditions. Other
  ionisation methods are available leading to the
  stabilisation of pseudo molecular ions, negative
  ions and adduct ions. Many methods employ
  energetic electrons to initiate the ionisation
  primary ionisation. However, if a reagent
  (reactant) gas is introduced a reaction of the
  primary ion occurs and chemical ionisation
  results as a secondary process leading to the
  production of a secondary ion from molecules of
  the sample to be analysed. Ionisation can be
  produced electrically (ESI) Atoms and ions can
  also be used to ionise molecules under soft
  ionisation conditions (FAB). Lasers can be used
  to desorb and ionise molecules (MALDI)
• Chemical ionisation positive ion CI Chemical
  ionisation occurs when a reagent gas such CH4,
  NH3 or isopropane is introduced into the ion
  source at low pressure (lt 1 torr) ionisation
  occurs in the reagent gas to produce positive
  ions characteristic of the reagent gas
• CH4 e- ? CH4 2e- CH4 CH4 ? CH3.
  CH5 M CH5 ? MH CH4
• The positive ion produced because of its high
  acidity will protonate a neutral molecule of the
  sample introduced to generate a pseudo-molecular
  ion (M H) characteristic of the compound
  analysed. If this ion is produced in an
  energetic state, excess internal energy imparted
  during protonation, as is the case with methane,
  it will decompose leading to lower m/z fragment
  ions. However, if a softer chemical ionisation
  is employed as with NH3 or isopropane , the
  pseudo molecular ion will be more stable. NH3
  leads to the formation of adduct ions at m/z, (M
  18) and (M 35) but only with molecules
  containing heteroatoms with lone pairs, such as O
  and N.

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• Ionisation methods continued
• Chemical ionisation Negative ion CI NICI can
  be achieved in several ways. When a reagent gas
  is introduced the electrons emitted from the
  filament ionise this gas producing more electrons
  which have lower energies brought about through
  inelastic collisions with neutral molecules.
  Thermal electrons are subsequently produced when
  the electron energy is reduced to that of the
  surroundings and these electrons undergo
  resonance capture reaction with molecules of a
  compound to be analysed having a finite electron
  affinity. The process of electron capture is
  simply as follows
• M e-t ? M.- ? A- B.
• The radical anion produced is sometimes unstable
  and undergoes dissociative electron capture
  resulting in the formation of stabilised A-
  characteristic of the molecule. Little
  fragmentation often occurs in NICI.
• NICI can also be conducted by using a reactant
  gas, X, from which reactant gas ions, X-, are
  generated on interaction with electrons produced
  by ionisation of the reactant gas. The reactant
  gas ion undergoes charge transfer with a molecule
  of a compound introduced frequently by proton
  transfer X- M ? XH (M H)- .
• Fast atom bombardment and secondary ion MS The
  molecular ion generated through electron
  bombardment is very frequently unstable because
  the molecule is sensitive to breakdown. A much
  softer ionisation must be employed to produce
  stable molecular ions in this instance. FAB
  employs fast atoms or ions to sputter ions from
  molecules of the analyte in a liquid matrix, e.g.
  glycerol in which the solid compound is
  dispersed. The process is essentially one of
  desorption ionisation. Fast atoms are prepared by
  accelerating xenon (or argon) ions to an energy
  in the range of 6 9 KeV and then neutralising
  these ions by charge exchange (electron transfer)
  as they pass almost stationary xenon atoms at low
  pressures, the excess kinetic energy is
  transferred to the xenon atoms formed.

25
? Xe Xe ? Xe Xe

• When the fast atoms impart energy into the
  solution of the sample in the matrix molecules of
  the compound are desorbed, often as ions, by
  momentum transfer. FAB has the capability to
  analyse large polar molecules and it is
  beneficial to couple a FAB source to magnetic
  sector instrument having a mass range of up to
  10, 000 12, 000 daltons at full accelerating
  voltage. An alternative matrix is
  thioglycerol/diglyerol (1/1). It is important
  that the sample dissolves in the matrix and is
  marginally more hydrophobic than the matrix thus
  occupying the matrix/vacuum interface. Cs ions
  are also employed in FAB analysis (see schematic
  diagram - 10).
• Positive and negative ion FAB mass
  spectra can be recorded, MH and (M-H)- the
  molecular ion species are usually the most
  abundant ions and since large molecules can be
  analysed RMM determination is extremely easy by
  these techniques.
• Plasma desorption The principle of plasma
  desorption employing 252Cf PD source is
  illustrated in the diagram (see schematic diagram
  - 11). The sample is deposited on a thin metal
  foil, usually nickel. Spontaneous fission of
  252Cf occurs and each fission event results in
  the formation of two fragments travelling
  diametrically at 180o (momentum conservation). A
  typical pair of fragments is 142Ba18 and
  106Tc22 with kinetic energies of roughly 79 and
  104 Mev, respectively. When a high-energy
  fission fragment passes through the sample foil,
  extremely localised heating occurs, producing a
  temperature in the region of 10,000 K. The
  molecules in this plasma zone are desorbed with
  the production of both positive and negative
  ions. The ions formed may be accelerated out
  into the analyser system.
• 252Cf PD is more able to produce
  molecular ion signals than FAB or SIMS in the RMM
  range 10,000 20,000 daltons, where the
  precision of mass determination may be /- 10
  20 mass units.

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5. MALDI-MS Matrix-Assisted Laser Desorption
Ionisation TOF.MS is an alternative to PD-MS, see
Diagram 12. For this technique a matrix is
required in which the target compound is
dissolved usually a viscous organic liquid
glycerol, cinnamic acid, nitrophenol derivative
Since lasers only generate coherent light at
specific wavelengths not all compounds undergo
MALDI. The firing of the laser is synchronised
with the TOF to enable accurate flight times and
m/z to be recorded.
27
Diagram 10 FAB mechanism
28
Diagram 11 Plasma Desorption MS
29
Diagram 12 MALDI MS
30

• Ionisation methods and interfaces for
  chromatography
• Ionisation devices have been specially designed
  for applications with chromatography in addition
  to the universally used EI and CI mode (stand
  alone MS). The development has been rapid and GC
  was first coupled to mass spectrometry via a jet
  separator for packed column analysis and by
  direct insert for capillary column mode.
  Whereas, for LC applications the advances were
  more recent and have been slower, progressing
  through more design stages Moving belt,
  Particle Beam, (FAB also stand alone),
  Thermospray, PD, Atmospheric Pressure Chemical
  Ionisation and API/Electrospray (originally
  developed in 1930s). Because the mobile phase is
  a liquid and a flow rate of 1 ml/min entering the
  MS on vaporisation, would create a volume of
  vapour which the mass spectrometer pumping system
  could not cope with. Therefore, there is a need
  in the design of the interface to reduce the
  volume of vapour entering.
• The directly coupled interface for GC requires an
  interface oven heated to 300oC between the GC and
  MS through which the capillary column passes
  before entry into the ion source. The column exit
  will be at low pressure because of the vacuum
  effect of MS manifold reducing the column
  performance and shortening retention times. To
  overcome this effect the column can be connected
  via a split coupling to allow ambient pressure at
  the column exit by introducing helium gas via an
  auxiliary line (and cutting of the
  analytes/solvent eluting from the column if
  required). This device enables matching of
  retention data from GC/MS analysis to that
  obtained from conventional GC.
• Interfaces for LC The interfaces developed
  specifically for LC include MBI, PBI, TSP, APCI
  and ESI. The discussion will focus mainly on APCI
  and API/ESI. The interface can be divided into
  two types, ionisation interfaces, TSP and ESI and
  pre-ionisation interfaces, APCI (MBI and PBI)
  whereby ionisation occurs in the ion source
  region.

31

• Electrospray ionisation ESI is a technique used
  to produce gaseous ionised species from a liquid
  solution generated by creating a fine spray of
  charged molecules in the presence of a strong
  electric field. The charged molecules are
  sprayed from the tip of metal nozzle held at
  approximately 3 4 kV and then electrostatically
  directed towards the mass spectrometer inlet.
  Either gas, heat or a combination of both are
  applied to the droplets to induce vaporisation
  prior to entering the MS. As the solvent
  evaporates, the electric field density increases,
  hence the mutual Coulombic repulsion between
  individual species becomes so great that it
  exceeds the surface tension and at the Rayleigh
  limit the droplet explodes forming a series of
  smaller, lower charged droplets. Ions leave the
  droplet through what is known as a Taylor Cone.
  The process of shrinking is repeated until
  individually charged analyte ions are formed.
  Increasing the solvent evaporation, by
  introducing a drying gas flow counter current to
  the sprayed ions increases multiple charging.
• A Schematic diagram - 13 of an ESI Source and
• B diagram 14 demonstrating formation of a
  Taylor cone and ion ejection
• Atmospheric Pressure Ionisation can be carried
  out in two ways
• AP MS employing ESI which is termed, ionisation
  in the interface. To aid solvent evaporation and
  reduction in droplet size a dryng gas N2 is
  introduced prior to discharge also later as a
  curtain gas to reduce clustering of ions which is
  a feature of ESI.
• AP MS using chemical ionisation whereby a fine
  spray from a nebuliser is directed into the ion
  source to which an ionisation device is fitted,
  such as discharge needle (Corona) at 7 8 kV or
  plasma generated by 252Cf. Here also N2 is
  introduced to aid evaporation.
• The AP assists in reducing the flow rate of
  liquid eluting from the HPLC column from 0.5
  ml/min to 10 100 ?l/min so that the vacuum
  system of the MS can handle the inflow of the
  vapour generated from the liquid (NB. 1 ml of
  liquid, usually water, produces over 1000 ml
  vapour).

32
Diagram 13 Electrospray ion source
33
Diagram 14 ESI and formation of Taylor Cone
(coulombic explosion)
34

• Tandem (Hybrid) Mass Spectrometry for Multiple
  Modes of Scanning (MS-MS)
• T(H)MS is an instrumental technique in which
  multiple MS-MS modes of scanning can be
  conducted, is divided into three categories
  comprising, for the first two, combinations of
  analysers
• one type of mass spectrometer only EB-EB, BE-EB
  or Q-q-Q both pure tandem MS-MS (the lower case q
  denotes a transmission only quadrupole)
• two or more types of analyser in a hybrid
  configuration EBqQ Q-TOF EB-Q-TOF
• OR a single MS system
• Ion trap MS in which MSn can be conducted where n
  1 - 12.
• The tandem MS-MS involving E an B sector
  analysers has been largely superseded by 1. TMS
  involving Q analysers, 2. hybrid MS and 3. ion
  trap MS.
• The advantages of TMHS are several higher
  specificity for characterising a target compound
  in a complex mixture and higher sensitivity,
  particularly at higher mass resolution for the
  compound of interest (reduced ion background
  interference, higher S/N ratio).
• The TMS configuration, QqQ, for example, allows
  primary or reactant ions to be selected in the
  first Q, to undergo collision-induced
  deactivation (CID) in second Q (collision gas
  argon transmission only) and the product ions
  resulting to be analysed by dynamic mass scanning
  of the third Q (see type (i) below). For a full
  demo of the operation for MS-MS see Applied
  BioSystems HPLC TSQ MSppt notes.
• The scanning can be of three types
• Static mass scanning of the first Q and dynamic
  mass or selected mass scanning of the third Q
  (reactant ions fragmenting to the product
  ions
• Dynamic mass scanning of the first Q and static
  mass scanning of the third Q (product ions
  having the same reactant ion)
• Static mass scanning of the first Q and static
  offset mass scanning of the third Q to monitor
  constant neutral loss (mass difference between
  reactant ion 1 and product ion 2).

35

• Detectors
• There are several types of detector suitable for
  use with MS
• Electron and Photomultipliers The EM relies on
  a conversion electrode (dynode) at high voltage
  ( 1 to 3 kV range), at which a positive ion is
  transformed into an electron, an electron gain
  multiplication factor, 106, pre-amplification of
  the electron current, conversion of the electron
  current into an analogue voltage prior to
  digitisation via a fast ADC interfaced to the
  Data System. The modern EM is of a continuous
  (trumpet) dynode type. channeltron c oate on
  the inside with an electron emissive material).
• see Schematic Diagram - 15
• The detection in the PM is brought about by
  transformation of a positive ion at a conversion
  dynode into an electron that subsequently
  impinges on a phosphor screen producing a light
  photon (h?), followed by multiplication,
  pre-amplification and current-voltage conversion
  as indicated above.
• The detection and conversion of a negative ion
  is complicated by the fact that the first dynode
  of the EM cannot be used to directly detect the
  negative ion. A further conversion dynode (at
  high positive kV) is added to transform the
  negative ion into a positive ion which in turn is
  converted by a dynode at -kV into an electron
  prior to multiplication, pre-amplification and
  current to voltage conversion.
• see Schematic Diagram - 16
• Microchannel plate This detector consists of an
  array of glass capillaries (10 25 ?m i.d.) that
  area coated on the inside with an electron
  emissive material. The capillaries are biased at
  a high voltage, and like the channeltron, an ion
  that strikes the inner wall of one of the
  capillaries creates a cascade of secondary
  electrons. This cascading effect creates a gain
  of 104 and produces a current at the output.

36
Schematic Diagram - 15
37
Schematic Diagram - 16
Schematic Diagram - 16
38

• Data Systems
• Data system electronically interfaced to MS is
  vitally important for
• Control of MS modes of scanning, voltages,
  currents, etc., tuning and mass calibration
• data acquisition the digital voltage signal
  response for each ion scanned transmitted from
  the fast ADC
• data processing to obtain mass spectra, RA
  abundance lists, elemental composition, selected
  mass chromatograms, library search NBS/Wiley,
  quantitation (total ion chromatogram, TIC)
• Following tuning to optimise the response mass
  peak shape/intensity using a calibrant gas
  pftba (quadrupole) or pfk (EB or BE) and (PEG
  oligomers and Cs ion clusters for high mass) and
  mass calibration, acquisition of data can be
  carried out in conjunction with chromatography
  (GC or LC) or solid probe with ionisation modes
  - EI, FD/FI, CI, FAB, APCI/ESI, laser - LD/I,
  252Cf - PD
• Centroiding of the mass peak The digitised
  voltage and intensity of the signal (Im/z) of
  the ion peak detected (m/z) is proportional to
  the current carried by that ion and related to
  the time of appearance (tm/z) of that ion in the
  scan (a typical total scan time, span, is 0 - 1
  s). The calibration table stored on the DS is
  used to relate the tm/z to the mass (m/z) and
  thus the intensity v. m/z relationship can
  obtained to build up the mass spectrum from the
  molecular ion and all the fragment ions (m/z)
  detected for the compound being analysed. For
  accurately obtaining the mass of each ion
  detected a process of mass centroiding is
  require. An adequate sampling (data points per
  unit time) of the mass peak is required so that
  the centre of mass can be determined from the
  mass peak profile. The width of the mass peak
  decreases with resolution and thus the ADC
  sampling rate must be extremely fast (MHz range)
  for EB instruments.

see Schematic Diagrams 17 18
39
Schematic Diagram - 17
40
Schematic Diagram - 18
Schematic Diagram - 18
41

• Scan modes
• Full Scan For acquisition of the complete mass
  spectrum of a component in a complex mixture (GC
  or LC) or single compound on a probe the mode of
  scanning is full scan over the required mass
  range and 40 650 u is adequate for most organic
  compounds that are volatile (gas
  chromatographable). The mass range for a
  quadrupole is 1000 3000 u while that for a
  magnetic sector is dependent on the accelerating
  voltage.
• Selected ion monitoring/recording (SIM or SIR)
  For detection of low levels of a particular known
  target compound in a complex mixture, monitoring
  of a single ion, or selected ions characteristic
  of the compound is required. Accurate mass
  tuning and optimisation is required prior to SIM.
  The enhancement is proportional to the time of
  residence on the ion selected which is typically
  0.1 sec for a mass span of /- 0.02 u as compared
  to full scan, 1 - 2 msec for a mass span of 1 u.
  The employment of internal standards (13C or 2H
  labelled analogues) assists in the quantitation
  of target compounds. For EB or BE instruments a
  device to lock the mass selected for the standard
  to that of the target is required and accurate
  mass is carried by out by reference to the mass
  calibration gas (pfk)
• Multiple or selected reaction monitoring (MRM)
  This mode of scanning is possible only for tandem
  MS and similar to above the MRM can be general
  used or specifically to detect low levels of a
  target compound in a complex mixture by selecting
  the reactant ion and product ions generated, in
  one or several targets to be analysed, such that
  the CID is operated in a unique fashion.

42
Examples of Data Processing

• Total ion chromatogram (TIC), sum of all the ions
  detected during a chromatographic run in each GC
  peak and the background (chemical noise). Diagram
  19 20
• Mass Spectrum, raw and refined, RA of each m/z
  with respect to the base ion m/z (100). Diagram
  21
• Relative Abundance Mass Lists, raw refined, RA
  of an ion, m/z, relative to the base m/z (100).
• Mass Chromatogram / fragmentogram processing for
  location of target compounds in the TIC. using
  selected characteristic ions, m/z. Diagram 22
• Library search and match factors for raw and
  refined mass spectra to determine absolute,
  probable, possible and tentative ID.

Examples of Scan function operation

• Full Scan involves a selected mass range, 45
  450 u, depending on maximum m/z expected
  generating a Total Ion Chromatogram (TIC).
• Selected or Single Ion Monitoring (SIM/SIR)
  requires mass calibration and full scan
  chromatogram operation (TIC) to obtain the
  accurate masses of the ions to be monitored in
  the GC peak of the target compounds, usually the
  100 m/z and two diagnostic ions, prior to SIM
  chromatogram generation.
• Selected or Multiple Reaction Monitoring
  (SRM/MRM) in MS-MS involves scanning of
  characteristic precursor ions in Q1, CID in Q2
  and scanning of the product ions in Q3.
  Precursor ions for different compounds undergo
  different fragmentation reactions that may be
  compound specific. SIM can also be conducted
  using MS-MS.

43
Diagram 19 Typical MRM chromatogram
Desethylatrazine 188/146 2ng/mL
Atrazine 216/174 1ng/mL
Propazine 230/146 1ng/mL
Simazine 202/68 1ng/mL
Desisopropylatrazine 174/68 10ng/mL
API 2000 LC/MS/MS system with TurboIonSpray
source
44
Diagram 20 Single quadrupole vs. triple
quadrupole sensitivity/selectivity
analyte
analyte
MS
MS/MS
matrix
Injection of 8.0 ng/ml Acyclovir
much better detection limits in MS/MS due to
higher selectivity in MS-MS
45
Diagram 21 Typical Mass Spectra using MS/MS
Base Mass (ion) Peak, 100
Possible Molecular Mass (ion)
Intensity, or relative
Product Ion Scan 35V Haloperidol
100x more sensitivity in full scan
Mass/Charge Ratio, m/z
46
Diagram 22. Mass Chromatograms (Fragmentograms)
generated through Data Processing from the Total
Ion Chromatogram (m/z 191 and 218 selected),