PG3.51 Mass Spectrometry School of Chemistry

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PG3.51Mass Spectrometry School of Chemistry
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Overview

• Data acquisition
• HT
• Manual
• Ionisation
• LR
• HR
• High MWt
• Data analysis
• Web-based sample submission and data review

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High Throughput
Analysis time short reaction monitoring, fragile
samples Immediate access during working
day Multiple ionisation options to fit diverse
chemistries Generic methods not optimised to
your specific sample Sample overload should not
be an issue if correct amount submitted by user
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Manual
Method specific conditions optimised to specific
samples Correct ionisation method guaranteed Data
integrity assured interpretation not constrained
by pre-conceptions Slow dependent on staff and
instrument time
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Ionisation
Gas phase Electron Ionisation (EI) Generic
gas phase ionisation Information rich
spectra No molecular ion Chemical Ionisation
(CI) Gas phase basicity driven
ionisation Protonated molecule observed False
representation of sample Data more difficult to
interpret Condition dependent data
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Ionisation
Solution phase Electrospray Ionisation (ESI)
Simple data positive ion - protonated or
cationised molecule negative ion - deprotonated
molecule Requires basic or acidic site in the
molecule for ionisation Ionisation not
quantitative Atmospheric Pressure Chemical
Ionisation (APCI) Less restrictions on
functionality of molecule Not applicable to
large molecules Generic method
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Low Resolution MS
HT methods Compromised sample ionisation or
introduction GC interface competitive
ionisation for API
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High Resolution MS
Can resolve nominally isobaric species Accurate
mass - gives elemental composition of the ion
measured Is not equivalent to elemental
analysis Slow Different ESI source different
ionisation Not appropriate for impure
samples High mass cut-off of m/z 1000
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High Molecular Weight Molecules
Dedicated HT instruments Platform and
MALDI-TOF Needs in-depth training Good data
related to user knowledge Data analysis training
and processing time-consuming HT options are
generic methods Additives used compromise small
molecule analysis on Platform
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Data Analysis
Web based system consistent and correct data
processing View the data and interpret all ions
are there for a reason Time wasted reprocessing
can lead to misrepresentation of results
Impurities contaminants often ignored (these
can compromise ionisation) see www.soton.ac.uk/ms
web for info
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Web-based Sample Submission and Data Review

• Unique and secure single user accounts
• Networked computer within soton.ac.uk domain
• Save address in internet browser for access to
  RemoteAnalyzer
• http//152.78.196.1189999/RemoteAnalyzer/login.a
  spx
• Launch internet browser

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A current view of the mass spectrometer
Black Box
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ESI
MALDI
LC/MS
LC/MSMS
ESI
CZE/MS
ESI
Ion trap
TOF
QTOF
Quad.
FT-ICR
ESI
Waters
Bruker
AB-Sciex
Kratos
Thermo
Problem too many black boxes
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• In his book, Rays of Positive Electricity
  (1913), J.J.Thomson remarked
• I feel sure that there are many problems in
  chemistry which could be solved with far greater
  ease by this than by any other method. The
  method is surprisingly sensitive, ..requires an
  infinitesimal amount of material and does not
  require this to be specially purified.

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• How sensitive?
• Spectrum from 1 ng
• Actually possible from 10 pg
• Can use less
• 500 amol

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• Introduction
• What is a mass spectrometer?
• This is an instrument that operates under vacuum
  and separates charged gas phase species according
  to their mass-to-charge ratio.
• How does this help you?
• There are a variety of different types of mass
  spectrometer available. These employ various
  ionisation techniques and mass analysers that
  allow a wide range of molecules to be analysed
  from gases and small organic molecules through
  the mass range to large biopolymers.

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Chemistries studied

• Combichem
• Inorganic
• Natural product synthesis
• Bio-Organic
• Small Organic
• Peptides
• Proteins
• Polymers
• Biopolymers
• Organometallics

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Mass Spectrometry

• Instrumentation available
• VG Analytical 70-250-SE Normal geometry
  double focusing MS.
• Micromass TofSpec2E Reflectron
  MALDI-TOFMS.
• ThermoQuest TraceMS Single quadrupole
  GC-MS.
• Bruker ApexIII FT-ICR-MS.
• ThermoBioAnalysis Dynamo Linear MALDI-TOFMS.
• Micromass Platform II Open access single
  quadrupole MS.
• Waters ZMD Open access single quadrupole
  MS.
• ThermoQuest TraceMS Open access single
  quadrupole GC-MS.
• ThermoFinnigan LCQ Ion trap.
• Micromass PlatformLC Single quadrupole MS

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How do I determine the method of analysis?

• Consider which of the following is appropriate
• ionisation technique(s)
• sample introduction method
• analyser type and why
• why not other systems
• sample preparation
• what ions would you expect to see in the mass
  spectrum

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Introduction

• What is a mass spectrometer?

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What do I do?

• Which system do you use?
• Which ionisation process is appropriate?
• This is dependant on the nature of the sample
• ve or -ve ionisation?
• Again this is dependant on sample type

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Sample Introduction

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Multiuser Waters ZMD ES, APCI, FIA or
HPLC ThermoQuest TraceMS EI, CI, NICI
GC-MS Micromass Platform II ES,
APCI ThermoBioAnalysis Dynamo MALDI
online HPLC optional off-line infusion ES
MS optional off-line You must be trained by
either GJL or JMH
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MS only VG Analytical 70-250-SE LREI, LRCI,
LRFAB, HREI, HRCI, HRFAB HR-GC-MS. Micromass
TofSpec2E MALDI, maybe HRMALDI in future. Bruker
ApexIII ES, MSn, HPLC-MS, NS.
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How to submit samples for open access systems

• API
• 1 mg mL-1 in 1 mL of appropriate solvent,
  preferably MeCN or MeOH
• 2CV vial with teflon disk snap-cap top
• Microtitre plates (ZMD)
• EI/CI-GC-MS
• 10 mg mL-1 in 1 mL of appropriate solvent,
  preferably DCM
• 2CV vial with butyl septa crimptop

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How to submit samples for MS service

• Complete, in full, form and complete XL sheet in
  login area
• MS form available from website www.soton.ac.uk/ms
  web
• incomplete forms will be returned without analysis

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How to submit samples for MS service

• Fill in the sample progression sheet (XL)
• LR-MS (If open access is not appropriate)
• 1 mg of material in a screw-top vial clearly
  labelled, if in solution then state solvent and
  concentration hazard info
• HR-MS
• EI/CI 1mg of pure sample (accurately weighed),
  clearly labelled with weight recorded on vial.
• ES 10 µg mL-1 solution in MeOH or MeCN.
• LR-MS reference number info.

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• Simple Dos Donts
• Do not use any system unless you have been
  trained by GJL/JMH
• Label your bottle clearly, with a systematic
  reference number
• Remove your samples from the autosampler/Lab
  promptly after analysis
• Please spend time and clearly complete forms in
  full
• Do not hoard samples and submit in batches,
  especially HR at end of your final year
• If possible do not keep submitted samples in your
  labs
• Use the MS clinics, they are there to help you

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MS Clinics

• What are they?
• Daily help sessions between 11.30-12.00 and
  15.00-15.30
• How do you use them?
• Where appropriate, check data by reprocessing.
• Bring Spectra, MS file reference, structure,
  calculated mass and any other supporting
  information
• Come with a specific question

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• Simple Dos Donts

If in doubt ask us or E-mail msweb_at_soton.ac.uk
Julie Herniman John Langley 30 years
experience in MS Classically trained mass
spectrometrists Experience of MS vast array of
chemistries Departmental Commercial School
knowledge base for MS
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Atomic Structure
e-
e-
e-
Protons Neutrons
e-
e-
e-
Particle Mass, amu Charge Electron 0.00055
-1 Proton 1.0073 1 Neutron 1.0087 0
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Isotopes
mass number protons neutrons
C
C
12
13
6
6
atomic number protons
Atoms with the same atomic number but different
mass numbers are called isotopes.
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Atomic Weights
Natural Abundance
Mass, amu
Symbol
Carbon 12C 12.0000
98.89 13C
13.0033 1.11 Chlorine
35Cl 34.9689 75.77
37Cl 36.9659
24.23 Mass assigned as exactly 12
by international agreement

Atomic weight (C) 0.9889(12.0000)
0.0111(13.0033) 12.011 Atomic weight (Cl)
0.7577(34.9689) 0.2423(36.9659) 35.453
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Molecular Weights
Calculation of the MW of Reserpine, C33H40N2O9
Using Atomic Weights Using Single
Isotopes C 33 x 12.011 396.363 C
33 x 12.0000 396.000 H 40 x 1.0079
40.316 H 40 x 1.0078 40.312 N
2 x 14.0067 28.013 N 2 x 14.0031
28.006 O 9 x 15.9994 143.995 O
9 x 15.9949 143.954
608.687
608.272
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Isotope Ratios
C1 C10 C100
m/z R.I. X 100 X1 110 X2
60 X3 22
m/z R.I. X 100 X1 1.1
m/z R.I. X 100 X1 11.0
R.I.
m/z
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Isotope Ratios, cont.
Cl1 Cl2 Cl3
m/z R.I. X 100 X2 97.5 X4
31.7 X6 3.4
m/z R.I. X 100 X2 32.5
m/z R.I. X 100 X2 65 X4 10.6
R.I.
m/z
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Isotope Ratios, cont.
Br1 Br2 Br3
m/z R.I. X 34 X2 100 X4
98 X6 32
m/z R.I. X 100 X2 98
m/z R.I. X 51 X2 100 X4 49
R.I.
m/z
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• Questions
• What is meant by resolution?
• The ability of a mass spectrometer to
  distinguish between ions of different mass to
  charge ratio values is termed as resolution. A
  mass spectrometer has the ability to measure
  isotopic masses, e.g. the mass spectrum of HCl
  will show two peaks, nominally at 36 Da and 38 Da
  at an approximate ratio of 31, not at the
  average chemical mass of 36.5.

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36.5
50

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What is meant by isotopic mass, average mass
and nominal mass?

• Consider a given empirical formula, e.g.
  C60H122N20O16S2.
• The average mass, calculated using the average
  atomic mass 1443.8857 Da.
• The monoisotopic mass, calculated using the
  exact mass of the most abundant isotopes
  1442.8788 Da.
• The nominal mass, calculated using the integer
  mass of the most abundant isotopes 1442 Da.
• Which mass do you use?
• This depends on the resolving power of the MS
  and the mass of your molecule in general for
  small molecules use isotopic masses.

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• Introduction
• What is a mass spectrometer?

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• Analysers
• Sector Instruments
• Quadrupole Analysers
• Time-of-Fight (TOF)
• Ion Traps

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• Analysers
• Sector Instruments
• Original instruments based on fixed field
  magnets. Introduction of second sector, the
  electrostatic analyser and the electromagnet
  increased the resolution and mass range of the
  instruments. Magnet sector machines operate as
  low resolution and high resolution instruments
  and can be constructed as either normal geometry
  or reverse geometry machines. The configuration
  affects the ultimate resolution and also the MSMS
  capabilities of the instrument. Sector machine
  operate with high voltages on the ion sources,
  commonly 8kV.

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• Analysers
• Quadrupole Analysers
• Four concentric rods wired in pairs with
  alternating rf dc voltages applied. Ions that
  undergo stable oscillations are separated
  according to their m/z ratio and detected. The
  majority of quad systems are low resolution
  instruments they do not operate at high voltages
  and because analysis is achieved by switching of
  voltages is capable of fast scanning and
  consequently are ideally suited to interfacing to
  GC, HPLC, CEC and CE.

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• Analysers
• Time-of-Fight (TOF)
• Probably the simplest type of mass analyser. As
  the name suggests it measure the time it takes
  an ion to travel a specific distance. The ion is
  initially accelerated by a high potential
  20-30kV, different mass ions will be accelerated
  into the analyser with initial velocities
  proportional to their masses and subsequently
  arrive at the detector at different times. The
  advent of DE (PE, TLF) has improved the
  resolution of these systems. Similarly
  reflectron systems produce higher resolution data
  than linear systems. ES-TOFs, GC-TOFs and
  hybrid instruments have recently become available.

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• Analysers
• Ion Traps
• The ion trap functions both as an analyser and
  an ion storage device. The ions are produced or
  introduced into the ion trap, trapped and can be
  progressively ejected form the ion trap volume
  by ramping of the rf voltage. The trapped ion
  can also be induced to undergo dissociation and
  hence produce the MSMS spectrum within the one
  analyser. Note the difference with FT-ICR-MS
  where the frequency of the circulating ions is
  measured and Fourier Transformed in give the MS.

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Mass 28 on a quad - unit resolution

100
28.00619
90
80
70
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Intensity (age)
50
40
30
20
10
0
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Low resolution on a sector - R 1000

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High resolution on a sector - R 10 000

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Comparison in ion profiles

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MS HV scan _at_ R 10000 (uncalibrated)

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• Introduction
• What is a mass spectrometer?

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Ionisation Techniques

• Electrospray (ESI)
• Electron Ionisation (EI)
• Chemical Ionisation (CI)
• Matrix-Assisted Laser Desorption/Ionisation
  (MALDI)
• Atmospheric Pressure Chemical Ionisation (APCI)
• Fast Atom Bombardment (FAB)
• Others

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Electrospray ionisation source
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• Electrospray Ionisation (ESI)
• This is a very mild, solution based ionisation
  process. It relies on the analyte having either
  a basic site or acidic site for ionisation. The
  eluent is sprayed at a high electropotential.
• M (sol) ? (M nH)n
• In the case of singly charged ions signals are
  observe for the protonated molecule or cationised
  molecule
• (M X) where X H, NH4, Na, K.
• For larger molecules then multiple charging can
  take place. Note that within the multiple charge
  envelope the charge states will be incremental,
  i.e. vary by 1. The true mass of the molecule
  can be obtained by solving the simultaneous
  equations or by using one of the many
  deconvolution packages available with the
  instruments.

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Electrospray ionisation process
M (sol) ? (M nX)n (gas phase) M (sol) ?
(M - nX)n- (gas phase) where X H, NH4, Na, K.
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Electron ionisation process

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• Electron Ionisation (EI)
• Ionisation by electron bombardment on a
  gas-phase molecule is an inelastic process the
  ionising electron loses some of its kinetic
  energy to the molecule, part of which is carried
  of by the secondary electron released on
  ionisation, and part of which is retained by the
  newly formed ion as internal energy. In a
  population of molecules a distribution of
  energies is transferred, as illustrated in the
  figure. The ionisation energy (IE) is the minimum
  energy required to ionise a molecule, typically
  about 8 - 12 eV for most organic compounds, so
  molecules acquiring energies greater than IE will
  be ionised. The appearance energy is the minimum
  energy needed for ionisation and reaction to form
  a fragment ion, and for a simple bondbreaking
  reaction this may be 2 or 3 eV above the
  ionisation energy. Each possible reaction path
  will have its own characteristic AE.

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• Electron Ionisation (EI)
• Gas phase molecules are bombarded with a stream
  of electrons to produce a radical cation. This
  is a harsh ionisation technique.
• M (g) e ? M. (g) 2e
• The molecular ion (MI) can undergo fragmentation
• M. ? F R.
• M. ? F. nm
• The Fragment ions can also undergo
  fragmentation
• F. ? F1 R.
• F ? F1 nm

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• Stability of the Molecular Ion
• If the mass spectrum shows a strong molecular
  ion peak this means the ion can stabilise the
  charge effectively. This is often the case for
  aromatic compounds, which give large molecular
  peaks and few fragment ion peaks. Benzene has its
  base peak (100 relative abundance) at m/z 78
  there are no major fragment peaks, the molecular
  ion representing more than 50 of the total ion
  current.
• This is because ionization of benzene can be
  achieved by removal of an electron from the
  delocalized pi-system, without seriously
  weakening the structure, and the ion thus formed
  can delocalise the charge over the whole
  molecule.
• By contrast, ionisation of the saturated
  cycloalkane cyclohexane can only be through loss
  of a sigma-electron, leaving a weak C-C or C-H
  single-electron bond. The molecular ion at m/z 84
  is not the base peak, and it represents less than
  20 of the total ion current

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• Fragmentation and Fragment Ion Stability
• Electron ionisation mass spectrometric reactions
  occur at low pressure in the gas phase and are
  unimolecular. They can be sequential or they may
  occur in parallel.
• In a complex spectrum there will be a network of
  competing and consecutive reactions, but a simple
  spectrum such as that of acetone shows only three
  significant peaks and this may conform to one of
  these schemes.

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• Fragmentation and Fragment Ion Stability
• Chemical reactions are controlled by both
  thermodynamics and kinetics, and whether the
  majority of molecular ions break down to fragment
  ions will depend not only on their energy but
  also on the time available for reaction. This
  time is the period spent in the ion source, which
  is typically 1us. This is a long time in relation
  to bond vibrations and will be adequate for
  simple bond breaking reactions.
• However, complex rearrangements may require the
  ions to attain shapes that are difficult to
  achieve, and such reactions may take longer than
  1us. This means that some fragment ions formed by
  high energy pathways could be more abundant in
  the spectrum than other ions formed by low energy
  pathways the latter are dependent on a
  rearrangement.

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• Fragmentation and Fragment Ion Stability
• Energetically the McLafferty rearrangement is
  more favourable, and yet because of steric
  requirements make it slower than the simple bond
  breaking reaction. Many of the molecular ions
  will undergo the bond breaking reaction before
  they have had time to undergo the McLafferty
  rearrangement.
• It is frequently the case that rearrangement
  ions are favoured on energetic grounds as they
  are formed in processes that involve bond
  formation as well as bond breaking. Energy is
  required to break bonds, therefore energy is
  given back in the making of new bonds.
• The McLafferty rearrangement is just one of many
  types of rearrangements.

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• Fragmentation and Fragment Ion Stability
• In considering fragmentation mechanisms it is
  generally assumed that the site of the charge is
  located at a specific site in the ion, and that
  this will initiate the reaction. A complex
  molecule with more than one functional group may
  be ionised at a number of different sites, giving
  different types of ion and different types of
  reaction.
• The site of ionisation will then be reflected in
  the origin of the peaks in the mass spectra.

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Chemical ionisation process

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• Chemical Ionisation (CI)
• The reagent gas reacts exothermically with the
  molecule M, behaving as a strong Bronsted acid,
  giving the protonated molecule MH
• The proton affinities of methane and ammonia
  are respectively 551 and 854 kJ/mol so that the
  transfer of a proton from CH5 to an organic
  molecule is always quite exothermic, giving rise
  to appreciable fragmentation.
• For less basic substances, such as
  oxygen-containing compounds, on the other hand,
  the transfer of a proton from the NH4 ion to the
  sample molecule is endothermic and therefore will
  not occur. Instead, the reactants form an adduct
  which usuallv undergoes limited fragmentation.
• The analyte must have a greater proton affinity
  (gas phase) than the reagent gas ions for proton
  transfer to take place. In some cases adduct
  ions are formed for compound containing a large
  number of functional groups.

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• Chemical Ionisation (CI)
• A reagent gas is used and is bombarded with a
  stream of electrons to produce a radical cation.
  
• RG (g) e ? RG. (g) 2e
• The reagent gas MI, e.g. ammonia, undergoes
  collision with neutral reagent gas
• NH3. NH3 ? NH4 NH2.
• Proton transfer takes place between the
  protonated ammonia (in excess) and the analyte M
• NH4 M ? MH NH3
• With ammonia the adduct ions can also be formed
• NH4 M ? MNH4

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• Chemical Ionisation (CI)
• CI is a softer ionisation technique than EI,
  some fragmentation may occur.
• Note
• MNH4 - H2O has the same nominal mass as M
• Rarely do you see radical cations in CI spectra
  unless there is a problem with the gas supply or
  ion source.
• Only use CI if you do not observe the MI by EI.

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Chromatogram
Peak Retention Time
Spectrum
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MALDI
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MALDI
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MALDI
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• Matrix-Assisted Laser/Desorption Ionisation
  (MALDI)
• The sample is mixed with a UV absorbing matrix,
  the latter in great excess. Singly charged
  protonated and/or cationised molecules are
  produced, hence the need for a large mass range
  analyser. Also good for involatile small
  molecules, peptides, biopolymers and polymers.
  Used in conjunction with TOFs to give large mass
  range.
• Uses pmols of material.

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MALDI ionisation process?
M (solid) ? (M X) (gas phase) M (solid)
? (M -X)- (gas phase) where X H, NH4, Na, K.
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• MALDI Contamination Limits
• Analysis is relatively insensitive to
  contaminants.
• Phosphate 20 mM EDTA 1 mM
• Detergents 0.1 Glycine 20 mM
• Glycerol 2 Sodium Citrate 20 mM
• Buffer (Tris) 50 mM K phosphate 25 mM
• Guanidine 1 M Na phosphate 0.1M
• Na azide 1 Octyl glucoside 0.3
• SDS 0.05 Ammon. Bicarb. 0.1M
• Suggested concentrations
• 10 pmol _at_ lt10 000 Da (pure)
• 100 pmol _at_ gt50 000 Da (pure)

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• Atmospheric Pressure Chemical Ionisation (APCI)
• Very similar to ES, except that the spray is
  neutral and passes through a corona discharge.
  Singly charged protonated molecules are produced.
  Ionisation is not so dependent on the
  functionality of the analyte cf. ESI, though some
  functionality is required.

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• Ionisation Techniques
• EI CI are gas phase processes, therefore the
  sample must be volatile and thermally stable.
• ES, APCI MALDI are condensed phase techniques,
  therefore the samples must be soluble.
• E.g. MeCN, MeOH, H2O or mixtures of these.
• Do not use involatile buffers for spray
  techniques.

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• Now we can answer some questions
• Which ionisation process is appropriate?
• This is dependant on the nature of the sample.
• Functionality, volatility (MP/BP), size.
• ve or -ve ionisation?
• Again this is dependant on sample type.
• Functionality, pH of solution.

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• Determine the method of analysis
• Consider which of the following is appropriate
• ionisation technique(s)
• sample introduction method
• analyser type and why
• why not other systems
• sample preparation
• what ions would you expect to see in the mass
  spectrum

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• Ionisation Techniques
• EI CI are gas phase processes, therefore the
  sample must be volatile and thermally stable.
• For OA EI, CI GCMS rmm lt 500Da, MP lt 150 oC
• Solvents DCM, EA, cHexane. NOT aqueous.
• ES, APCI MALDI are condensed phase techniques,
  therefore the samples must be soluble.
• For OA ES APCI use MeCN, MeOH, H2O or mixtures
  of these.
• Do not use DMF, DMSO or THF, check with GJL or
  JMH if other solvents are used.
• Do not use involatile buffers.

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• Interpretation
• How do I interpret my spectra?
• Check the quality of the data does the total ion
  chromatogram look ok?
• Does the mass spectrum show significant peaks
  above the baseline?
• Identify any major peaks.
• Look for telltale isotope patterns.
• Remember the Nitrogen Rule. (The Nitrogen Rule
  A neutral compound containing an odd number of
  nitrogen atoms will always have an odd molecular
  mass.)
• Do the masses fit with the proposed structure?
• Have you used the appropriate masses in your
  calculations?

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• Interpretation
• How do I interpret my spectra?
• Identify the MI
• Check isotope pattern - halogens, sulphur, tin
  etc.
• Odd/Even Mass - Nitrogen Rule
• If EI check intensity of MI - aromatic or
  aliphatic
• Accurate mass - calculate r db
• CxHyNzOn
• r db x - 0.5y 0.5z 1
• whole number OE
• even number EE

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• Reporting Data
• State method, ionisation mode, e.g.
• LRMS (EI) m/z 220, 80 (M.) 205, 100
  (M-CH3)
• HRMS (ES) Found 501.1234 Da (M H),
  calculated 501.1238 Da
• You should report the Base Peak (BP), Molecular
  Ion (MI) or protonated or cationised molecular
  ion(s) (M X) and any other significant peaks.
  For EI spectra the eight major peaks are
  considered representative.
• Remember higher mass ions of low intensity can be
  more important that high intensity low mass ions.

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C H N O mass
DBE error . Mass Analysis for mass
304.1540660 1 17 22 1 4
304.1543346 7.5 8.832e-07 2 2 18
13 5 304.1548372 0.5 2.535e-06 3
15 20 4 3 304.1529919 8.0
3.531e-06 4 18 18 5 0
304.1556720 12.5 5.280e-06 5 4 20
10 6 304.1561798 0.0 6.950e-06 6
14 24 0 7 304.1516545 3.0
7.928e-06 7 13 18 7 2
304.1516493 8.5 7.946e-06 8 20 20
2 1 304.1570147 12.0 9.695e-06 9
5 16 14 2 304.1575172 5.0
1.135e-05 10 6 22 7 7
304.1575225 -0.5 1.136e-05   Mass
Analysis for mass 182.1176340 1 10 16
1 2 182.1175552 3.5 4.328e-07 2
8 14 4 1 182.1162125 4.0
7.805e-06 3 7 18 0 5
182.1148751 -1.0 1.515e-05 4 6 12
7 0 182.1148698 4.5 1.518e-05 5
5 16 3 4 182.1135324 -0.5
2.252e-05 6 0 12 11 1
182.1220805 0.5 2.442e-05 7 3 14
6 3 182.1121898 0.0 2.989e-05 8
2 14 8 2 182.1234232 0.0
3.179e-05 9 1 12 9 2
182.1108471 0.5 3.727e-05 10 4 16
5 3 182.1247658 -0.5 3.916e-05