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Mass Spectrometry. 2012 Fall version dslee@swu.ac.kr. Basic theory of mass spectrometry

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slide1

Mass Spectrometry

2012 Fall version

dslee@swu.ac.kr

slide2

Basic theory of mass spectrometry

The mass spectrometer(MS) is an instrument that serves for establishment of the molecular weight and structure of both inorganic and organic compounds, and the identification and determination of analytes in complex mixtures.

The MS is an instrument capable of producing a beam of ions from sample under investigation, separating these ions according to their mass-to charge (m/z) ratios and recording the relative abundances of the separated ion species as a mass spectrum. The ion-currents corresponding to the different species are amplified and either displayed on an oscilloscope or a chart-recorder, or are stored in a computer. The peak intensities are plotted as ordinates, in arbitrary units or normalized with respect to the most important peak, which is assigned a value of 100.

slide3

100

128

80

a

60

Relative abundance (%)

40

102

20

77

64

c

c

c

51

b

b

b

b

0

50

100

150

Mass to charge ratio

m/z

Mass spectrum of CO2. Note that the molecular ion appears at m/z = 44 (C = 12, O = 16). Frag-ment ions appear at m/z values of 28, 16, and 12. These correspond to CO+, O+, and C+, resp-ectively.

The mass spectrum of naphthalene with electron impact ionization by 70 eV electrons.

a, molecular ion and base peak(C10H+8, 100%);

b, 13C isotope peak;

c, fragment ion peaks.

,

slide4

Introduction to mass spectrometry

Mass spectrometers use the difference in mass-to-charge ratio (m/e) of ionized atoms or molecules to separate them from each other. Mass spectrometry is therefore useful for quantitation of atoms or molecules and also for determining chemical and structural information about molecules. Molecules have distinctive fragmentation patterns that provide structural information to identify structural components.

All commonly used mass analyzers use electric and magnetic fields to apply a force on charged particles (ions). The relationship between force, mass, and the applied fields can be summarized in Newton's second law and the Lorentz force law:

F = ma (Newton's second law)

F= e(E+ v× B) (Lorentz force law)

where F is the force applied to the ion, m is the mass of the ion, a is the acceleration, eis the ionic charge, E is the electric field , v B is the vector cross product of the ion velocity and the applied magnetic field.

slide5

From Newton's second law, it is apparent that the force causes an acceleration that is mass-dependent, and the Lorentz force law tells us that the applied force is also dependent on the ionic charge. Therefore, it should be understood that mass spectrometers separate ions according to their mass-to-charge ratio (m/z) rather than by their mass alone.

  • The general operation of a mass spectrometer is:
  • create gas-phase ions
  • separate the ions in space or time based on their mass-to-charge ratio
  • measure the quantity of ions of each mass-to-charge ratio
  • The ion separation power of a mass spectrometer is described by the resolution R, which is usually defined as: 
  • where m is the ion mass and delta m is the difference in mass between two resolvable peaks in a mass spectrum.
slide6

Mass Spectrometry

Theory

In mass spectrometry, a small sample of a chemical compound is vaporized, bombarded with high energy electrons to ionize the sample, and the ions produced are detected based on the charge to mass ratio of the ions.

Ionization process in mass spectrometry.

slide7

Several different types of ions are produced in this process. If the compound loses only one electron, then a molecular ion, having the same mass as the original compound, is produced. This molecular ion is called the M+ ion (and it gives us the exact molecular weight of the compound).

The stream of high energy electrons is sufficiently powerful to break chemical bonds in the molecule, producing a series of molecular fragments. These positively charged fragments are also detected by the instrument, producing the mass spectrum. Organic chemical compounds will fragment in very specific ways depending on what functional groups are present in the molecule. Analysis of the fragmentation pattern can lead to the determination of the structure of the molecule.

Fragments produced by benzamide.

slide8

the analyte (A-B-C) undergoing ionization and fragmentation

  • the charged fragments (A+ B+ C+) being separated by mass
  • the fragments which are focused on the mass filter's exit slit passing into the detector
  • and the charged ions being detected.
slide9

Instrumentation

Components of a mass spectrometry

1) Inlet system : introduce a very small amount of sample ( micromole or less) into the mass spectrometer, where its components are converted to gaseous ions.

2) Ion source : converts the components of a sample into ions by bombardment with electrons, ions, molecules, or photon.

3) Mass analyzer : separates the analyte ions according to their m/z ratios.

4) Detector : converts the beam of ions into an electrical signal(currents) ; the detector output can be displayed or stored, to yield the mass spectrum.

5) Electronics of power supply and control of the systems.

6) Vacuum systems : maintain low pressures ( 10–5 to 10–8 torr); rotary vacuum oil pump, diffusion pump, turbomolecular pump.

slide10

Sample

introduction

Interfacing

Ion Source

Analyzer

Detector

Data

System

  • EI
  • CI
  • FAB
  • MALDI
  • ESI
  • APCI
  • Thermospray
  • Particle Beam
  • Quadrupole
  • Sector
  • TOF
  • FT-ICR
  • Ion Trap
  • Library
  • GC
  • LC
  • SFC
  • IC
  • CE
  • DLI
  • ICP
  • Solid probe

Vacuum System:

DP or TMP

  • MS/MS – Q/TOF, TOF/TOF
  • HRMS
  • LRMS

Mass Spectrometer Components

10–5 ~ 10–8torr

slide12

Vacuum system

  • Oil rotary pump : 50 ~ 200 ml/min
  • Turbomolecular pump : no cooling water, 10 min.
  • Oil diffusion pump : cooling water, 40~50 min. 600 ~ 1000 l/sec
  • Vacuum in MS 10-6 to 10-5 Torr
  • - Increase sensitivity
  • - avoid ion-molecule reaction
  • - collision free ion trajectories
  • - avoid background interference
  • Vacuum System:
  • - Pumps must be maintained ; Ultra-clean ; Vacuum regulation and maintenance
slide13

High vacuum

Ion

Source

Mass

Analyzer

Detector

Data

system

Interface

to vacuum

High vacuum

Interface

to vacuum

Mass

Analyzer

Detector

Data

system

Ion

Source

MS Configuration for Ionization

EI

API, APCI, ESI

slide14

Sample introduction

For EI and CI there are four main options

Heated reservoir septum inlet for pure gases or volatile liquids. Basically this is a heated reservoir (~200oC) with a small restriction ‘bleed’ into the ion source. Sample is injected into the reservoir through a septum.

Direct insertion probe for volatile solids. Sample is loaded into a quartz tube at the end of a probe and inserted directly into the ion source. The end of the probe can then be heated, if required, up to temperatures in excess 400oC.

Gas chromatograph (GC) for volatile, non-polar mixtures. The mixture is injected into the top of the GC column the end of which is passed, via a heated interface, directly into the ion source. The components of the mixture separate out during their passage through the column and enter the ion source sequentially.

Particle Beam interface for semi-volatile compounds that are amenable to EI and CI. This is a useful LC/MS interface that can also be used for rapid introduction of samples by flow injection. Samples are dissolved in a suitable solvent and the solution is introduced into the mass spectrometer using a suitable (e.g. HPLC) pump. The liquid is nebulised with helium gas to form an aerosol of solvent droplets. The stream of liquid droplets passes through a desolvation chamber and then a series of nozzles and skimmers that remove the solvent and helium, allowing a stream of solid particles (the sample) to enter the EI/CI ion source.

slide15

Chromatographic inlet systems

A> Interfacing GC with MS

1. Molecular jet separator (all-glass) : Ryhage, 1966

2. Membrane separator : Llewellyn, 1966

3. Effusion separator : Watson-Bieman, 1964

4. Open split coupling

5. Capillary direct interface

B> LC-MS interface

1. Moving belt wire interface

2. Thermospray

3. Atmospheric pressure inlet (API)

4. Electrospray

5. Particle beam

6. Dynamic FAB

GC-MS : Thermal vaporization of the analyte, separation, transfer of analyte to the MS, ionization and detection

GC

Transfer line

MS

Injection

Separation

Ionization Ion filtering Detection

Transfer

LC-MS : Separation, nebulization of the analyte, transfer of ions to the MS, and detection

LC

Interface

MS

Injection

Separation

Transfer Ionization

Ion filtering Detection

slide16

Ionizer

In the GC-MS discussed in this introduction, the charged particles (ions) required for mass analysis are formed by Electron Impact (EI) Ionization.  The gas molecules exiting the GC are bombarded by a high-energy electron beam (70 eV).  An electron which strikes a molecule may impart enough energy to remove another electron from that molecule.  Methanol, for example, would undergo the following reaction in the ionizing region:         CH3OH + 1 e  CH3OH+. + 2 e note:  the symbols +. indicate that a radical cation (radical ion) was formed

EI Ionization usually produces singly charged ions containing one unpaired electron.  A charged molecule which remains intact is called the molecular ion.  Energy imparted by the electron impact and, more importantly, instability in a molecular ion can cause that ion to break into smaller pieces (fragments).  The methanol ion may fragment in various ways, with one fragment carrying the charge and one fragment remaining uncharged.  For example:          CH3OH+. (molecular ion)  CH2OH+(fragment ion) + H.(or)   CH3OH+. (molecular ion)  CH3+(fragment ion) + .OH

http://www.chem.arizona.edu/massspec/intro_html/intro.html

slide17

Ionization techniques

- EI (electron impact)

- CI (chemical ionization)

- FAB (fast atom bombardment)

- ESI (electrospray ionization)

- MALDI (matrix assisted laser desorption ionization)

- APCI (atmospheric pressure chemical ionization)

slide18

Ionizationmethod

TypicalAnalytes

SampleIntroduction

MassRange

MethodHighlights

Electron Impact (EI)

Relativelysmallvolatile

GC orliquid/solidprobe

to1,000Daltons

Hard methodversatileprovidesstructure info

Chemical Ionization (CI)

Relativelysmallvolatile

GC orliquid/solidprobe

to1,000Daltons

Soft methodmolecular ionpeak [M+H]+

Electrospray (ESI)

PeptidesProteinsnonvolatile

LiquidChromatographyor syringe

to200,000Daltons

Soft methodions oftenmultiplycharged

Fast Atom Bombardment (FAB)

CarbohydratesOrganometallicsPeptidesnonvolatile

Sample mixedin viscousmatrix

to6,000Daltons

Soft methodbut harderthan ESI orMALDI

Matrix Assisted Laser Desorption(MALDI)

PeptidesProteinsNucleotides

Sample mixedin solidmatrix

to500,000Daltons

Soft methodvery highmass

Sample introduction / ionization method:

slide19

Electron Ionization (EI) 

Theory

Electrons are produced by thermionic emission from a tungsten or rhenium filament. A typical filament current is 1x10-4Amps. These electrons leave the filament surface and are accelerated towards the ion source chamber which is held at a positive potential (equal to the accelerating voltage). The electrons acquire an energy equal to the voltage between the filament and the source chamber - typically 70 electron volts (70 eV). The electron trap is held at a fixed positive potential with respect to the source chamber. A proportion of the electron beam will strike the electron trap producing the trap current. This is used as a feedback circuit to stabilize the electron beam.

A permanent magnet is positioned across the ion chamber to produce a magnetic flux in parallel to the electron beam (see figure). This causes the electron beam to spiral from the filament to the trap, increasing the chance and efficiency of analyte ionization. Gaseous analyte molecules are introduced into the path of the electron beam where they may be ionized by electronic interactions with this electron beam. Ionization can also be brought about by direct impaction of an electron with an analyte molecule. The positive ion repeller voltage and the negative excitation voltage work together to produce an electric field in the source chamber such that ions will leave the source through the ion exit slit. The ions are directed through the various focussing and centering lenses and are focussed onto the source exit slit. The ions can then be mass analysed.

http://www-methods.ch.cam.ac.uk/meth/ms/theory/eims.html

slide20

Schematic of a Kratos Analytical Electron Ionization source (as used on the MS890).

http://www-methods.ch.cam.ac.uk/meth/ms/theory/eims.html

http://masspec.scripps.edu/information/intro/chapter3.html#3.2.1.

slide21

Mechanisms of ion formation

  • Consider the ionization of the analyte species AB:
  • AB + e-* A+ + B- + e-
  • AB + e-* A+ + B° + 2e-
  • AB + e-* [AB+°*] + 2e- followed by [AB+°*]  AB+°
  • AB + e-* [AB2+*]" + 3e- followed by [AB2+*]"  A+ + B+
  • - very low abundance
  • AH + e-* AH* + e-followed by AH* + AH  [AH+H]+ + A-
  • - 'self chemical ionization'
  • Notes:
  • Species with a * superscript are in high energy sates. Species with a ° superscript are radicals.
  • Species with a " superscript are short lived intermediates which are not seen in the spectra.
  • 1 and 2 are the highest abundance and are termed instantaneous fragmentation. This is the reason why EI is considered a hard ionization process.
  • 3 is fairly high abundance and is the process responsible for the molecular ion formation. Unfortunately the highly energetic radical intermediate [AB+°*] tends to undergo fragmentation (or rearrangement) as a stabilizing process, this is responsible for the lower mass fragment ions present in the spectra. 4 is a very low abundance process, but theoretically it can occur.
  • 5 is a process which can occur at higher pressures (self Chemical Ionization), this is especially problematic in the ionization of alcohols and amines where you may find that the dominant ionization process is proton exchange between two analyte molecules, leading to the formation of the [M+H]+ pseudo-molecular ion.
slide22

EI

M + e– M+· + e – + e –

70 eV Molecular ion ~ 55 eV 0.1 eV

Ionization energies of valence electrons in formaldehyde.

Here are other first ionization energies:

CH3CH2CH2CH3 10.6 eV (sigma)

CH2=CHCH2CH3 9.6 eV (pi)

(CH3CH2)2O 9.6 eV (nonbonding)

C5H10NH 8.6 eV (nonbonding)

C6H6 9.2 eV (pi)

slide24

The Molecular Ion Peak

In the example above, involving benzamide (C7H7NO), the molecular ion (M+) peak had a mass to charge ratio (m/e) of 121. This value is calculated using the most abundant isotopes of the elements present in the molecule:7 × 12C = 84 7 ×1H = 7

1 ×14N = 14

1 ×16O = 16

Total 121

This type of calculation is called the unit mass molecular ion. Please refer to the spectrum for benzamide.

Mass spectrum for benzamide.

slide25

The observed abundance of the suspected molecular ion peak must correspond to expectations based on the assumed molecular structure. For example, highly branched substances undergo fragmentation readily; therefore, these types of substances will not have a very intense molecular ion peak. The intensity of molecular ion peaks will usually correspond to the following sequence:

aromatic compounds > conjugated alkenes > alicyclic compounds > sulfides > unbranched hydrocarbons > mercaptans > ketones > amines > esters > ethers > carboxylic acids > branched hydrocarbons > alcohols

For instance, if an unknown compound is suspected to be an alcohol, then the molecular ion peak will be quite small or completely absent.

slide26

Interpreting spectra

A simple spectrum, that of methanol, is shown here.   CH3OH+. (the molecular ion) and fragment ions appear in this spectrum.  Major peaks are shown in the table next to the spectrum.   The x-axis of this bar graph is the increasing m/z ratio.  The y-axis is the relative abundance of each ion, which is related to the number of times an ion of that m/z ratio strikes the detector.  Assignment of relative abundance begins by assigning the most abundant ion a relative abundance of 100% (CH2OH+ in this spectrum).  All other ions are shown as a percentage of that most abundant ion.  For example, there is approximately 64% of the ion CHO+ compared with the ion CH2OH+ in this spectrum.  The y-axis may also be shown as abundance (not relative).  Relative abundance is a way to directly compare spectra produced at different times or using different instruments.

EI ionization introduces a great deal of energy into molecules.  It is known as a "hard" ionization method.  This is very good for producing fragments which generate information about the structure of the compound, but quite often the molecular ion does not appear or is a smaller peak in the spectrum.

Of course, real analyses are performed on compounds far more complicated than methanol.  Spectra interpretation can become complicated as initial fragments undergo further fragmentation, and as rearrangements occur.  However, a wealth of information is contained in a mass spectrum and much can be determined using basic organic chemistry "common sense".

slide28

Following is some general information which will aid EI mass spectra interpretation:

Molecular ion (M .+):If the molecular ion appears, it will be the highest mass in an EI spectrum (except for isotope peaks discussed below).  This peak will represent the molecular weight of the compound.  Its appearance depends on the stability of the compound.  Double bonds, cyclic structures and aromatic rings stabilize the molecular ion and increase the probability of its appearance.

Reference Spectra: Mass spectral patterns are reproducible.  The mass spectra of many compounds have been published and may be used to identify unknowns.  Instrument computers generally contain spectral libraries which can be searched for matches.

Fragmentation:General rules of fragmentation exist and are helpful to predict or interpret the fragmentation pattern produced by a compound.  Functional groups and overall structure determine how some portions of molecules will resist fragmenting, while other portions will fragment easily.  A detailed discussion of those rules is beyond the scope of this introduction, and further information may be found in your organic textbook or in mass spectrometry reference books.  A few brief examples by functional group are described (see examples).

Isotopes:Isotopes occur in compounds analyzed by mass spectrometry in the same abundances that they occur in nature.  A few of the isotopes commonly encountered in the analyses of organic compounds are below along with an example of how they can aid in peak identification.

slide29

http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/MassSpec/masspec1.htmhttp://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/MassSpec/masspec1.htm

slide31

Ions with no nitrogenor an even # N atoms

odd-electron ionseven-number mass

even-electron ionsodd-number mass

Ions having anodd # N atoms

odd-electron ionsodd-number mass

even-electron ionseven-number mass

Mass Spectrometry

The Nitrogen Rule

If a compound contains an even number of nitrogen atoms (or no nitrogen atoms), its molecular ion will appear at an even mass number. If, however, a compound contains an odd number of nitrogen atoms, then its molecular ion will appear at an odd mass value. This rule is very useful for determining the nitrogen content of an unknown compound.

In the case of benzamide, the molecular ion appears at m/e = 121, indicating an odd number of nitrogen atoms in the structure.

The masses of molecular and fragment ions also reflect the electron count, depending on the number of nitrogen atoms in the species.

slide32

4-methyl-3-pentene-2-one has no nitrogen so the mass of the molecular ion (m/z = 98) is an even number

N,N-diethylmethylamine has one nitrogen and its molecular mass (m/z = 87) is an odd number. A majority of the fragment ions have even-numbered masses (ions at m/z = 30, 42, 56 & 58 are not labeled), and are even-electron nitrogen cations.

slide33

Electron ionization (70 eV) mass spectra of molecular ion region of benzene (C6H6) and biphenyl (C12H10).

Intensity of M+1 relative to molecular ion for CnHm :

Intensity = n × 1.08% + m × 0.012%

Contribution from 13CContribution from 2H

slide34

Methyl Bromide: An example of how isotopes can aid in peak identification.

The ratio of peaks containing 79Br and its isotope 81Br (100/98) confirms the presence of bromine in the compound.

slide36

Element

Isotope

RelativeAbundance

Isotope

RelativeAbundance

Isotope

RelativeAbundance

Carbon

12C

100

13C

1.11

Hydrogen

1H

100

2H

.016

Nitrogen

14N

100

15N

.38

Oxygen

16O

100

17O

.04

18O

.20

Sulfur

32S

100

33S

.78

34S

4.40

Chlorine

35Cl

100

 37Cl

32.5

Bromine

79Br

100

81Br

98.0

Relative Isotope Abundance of Common Elements:

slide37

Electron ionization mass spectrum (70 eV) of 1-bromobutane.

Mass spectrum showing natural isotopes of Pb observed as an impurity of brass.

slide38

Molecular Mass and Nominal Mass

The unit of atomic mass is the dalton, Da, defined as 1/12 of the mass of 12C. Mass specrometrists perfer the symbol “u” for “unified atomic mass unit”. “Da” and “u” are synonymous.

Atomic mass is the weighted average of the masses of the isotopes of an element.

Ex. Bromine 79Br 78.91834 Da 50.69%

81Br 80.91629 Da 49.31%

Br = 78.91834 × 0.5069 + 80.91629 × 0.4931

= 79.904 Da

The molecular Mass of a molecule or ion is the sum of atomic masses listed in a periodic table.

Ex. C2H5Br = 12.0107 × 2 + 1.00794 × 5 + 79.904 × 1 = 108.965

The nominal mass of a molecule or ion is the the integer mass of the species with the most abundant isotope of each of the constituent atoms.

Ex. C2H5Br = 12× 2 + 1× 5 + 79× 1 = 108

slide39

Chemical ionization (CI)

CI uses a reagent ion to react with the analyte molecules to form ions by either a proton or hydride transfer:

MH + C2H5+ MH2+ + C2H4

MH + C2H5+ M+ + C2H6

The reagent ions are produced by introducing a large excess of methane (relative to the analyte) into an electron impact (EI) ion source. Electron collisions produce CH4+ and CH3+ which further react with methane to form CH5+ and C2H5+:

CH4+ + CH4 CH5+ + CH3

CH3+ + CH4 C2H5+ + H2

slide40

Chemical ionization uses ion-molecule reactions to produce ions from the analyte. The chemical ionization process begins when a reagent gas such as methane, isobutane, or ammonia is ionized by electron impact. A high reagent gas pressure (or long reaction time) results in ion-molecule reactions between the reagent gas ions and reagent gas neutrals. Some of the products of these ion-molecule reactions can react with the analyte molecules to produce analyte ions.

Example

(R = reagent, S = sample, e = electron, . = radical electron , H = hydrogen):

R + e  R+.+ 2e

R+. + RH  RH+ + R.

RH+ + S  SH+ + R

(of course, other reactions can occur)

slide41

CI Reagent Gases

  • Methane:
  • good for most organic compounds
  • usually produces [M+H]+, [M+CH3]+ and [M+C3H5]+ adducts
  • adducts are not always abundant
  • extensive fragmentation
  • Isobutane:
  • usually produces [M+H]+, [M+C4H9]+ adducts and some fragmentation
  • A dducts are relatively more abundant than for methane CI
  • not as universal as methane
  • Ammonia:
  • fragmentation virtually absent
  • polar compounds produce [M+NH4]+ adducts
  • basic compounds produce [M+H]+ adducts
  • non-polar and non-basic compounds are not ionized
slide43

Mass spectra of the sedative pentobarbital, using EI (left) or CI (right).

The molecular ion ( m/z 226) is not evident with EI. The dominant ion from CI is MH+(protonated molecule). The peak at m/z 255 in the CI spectrum is from M(C2H5)+.

slide44

Fast Atom Bombardment (FAB)/Liquid Secondary Ionisation (LSIMS)

This technique, developed in the early 1980s, revolutionised the range of compounds analysable by mass spectrometry and opened up the field to most areas of biomedical research. Although now considered insensitive by comparison with more recently introduced ionisation modes, FAB still has a role as a rapid, reliable and robust technique for samples where quantity and purity are not a problem.

The sample is first dissolved in a liquid matrix. This is typically a viscous, low vapour pressure liquid such as glycerol or 3-nitrobenzyl alcohol. A few micro-litres of this liquid are placed on a small metal target at the end of a probe which is inserted into the mass spectrometer.

The liquid surface is then bombarded with a beam of high kinetic energy atoms (xenon or argon) or ions (caesium). Molecules are sputtered from the surface, enter the gas phase and ionise, either by protonation or deprotonation.

The resulting ions tend to be stable and exhibit little fragmentation.

slide45

Theory

The techniques of FAB and LSIMS involve the bombardment of a solid analyte + matrix mixture by a fast particle beam (see figure). The matrix is a small organic species (glycerol or 3-nitrobenzyl alcohol, 3-NBA) which is used to keep a 'fresh' homogeneous surface for bombardment, thus extending the spectral lifetime and enhancing sensitivity. In FAB, the particle beam is a neutral inert gas, typically Ar or Xe, at bombardment energies of 4-10 KeV, whereas in LSIMS, the particle beam is an ion, typically Cs+, at bombardment energies of 2-30KeV.

The particle beam is incident at the analyte surface, where it transfers much of its energy to the surroundings, setting up momentary collisions and disruptions. Some species are ejected off the surface as positive and negative ions by this process, and these 'sputtered' or secondary ions are then extracted from the source and analysed by the mass spectrometer. The polarity of the source extraction can be switched depending on what species are to be analysed. In the figure, the extraction is negative and thus only the positive ions are being analysed, this is termed +FAB. For LSIMS the atom beam is replaced by an ion beam.

Both FAB and LSIMS are comparatively soft ionization techniques, and are thus well suited to the analysis of low volatility species, typically producing large peaks for the pseudo-molecular ion species [M+H]+ and [M–H]-, along with structurally significant fragment ions and some higher mass cluster ions and dimers.

slide46

Schematic of a Fast Atom Bombardment source. The figure shows a schematic representation of a fast atom bombardment ion source, operating in the positive ion mode. The fast atom beam is incident at the analyte+matrix surface where it causes the 'sputtering' off of secondary ions. The positive ions are then extracted from the source into the mass spectrometer.

slide48

Matrix-assisted Laser Desorption/Ionization (MALDI)

Unlike FAB/LSIMS, this process uses a crystalline, rather than liquid, matrix, and a beam of photons, rather than atoms or ions. The net result is a dramatic increase, compared with FAB, in both sensitivity and mass range of analysable compounds. The sample is dissolved in a matrix and is allowed to crystallise on a stainless steel target. Suitable matrices include 2, 5 -dihydroxybenzoic acid and sinapinic acid. The target is then inserted into the mass spectrometer and the surface bombarded with a pulsed laser beam (typically generated by inexpensive nitrogen lasers with a beam wavelength of 337nm). Molecules are desorbed from the surface and ionise, usually by protonation or deprotonation. Any fragment or multiply charged ions are generally of low abundance in this ionisation mode.

slide49

(i) The Formation of a 'Solid Solution'. The analyte molecules are distributed throughout the matrix so that they are completely isolated from one other. This is necessary if the matrix is to form a homogenous 'solid solution' (any liquid solvent(s) used in preparation of the solution are removed when the mixture is dried before analysis).

(ii) Matrix Excitation. Some of the laser energy incident on the solid solution is absorbed by the matrix, causing rapid vibrational excitation, bringing about localized disintegration of the solid solution, forming clusters made up of a single analyte molecule surrounded by neutral and excited matrix molecules. The matrix molecules evaporate away from these clusters to leave the excited analyte molecule.

(iii) Analyte Ionization. The analyte molecules can become ionized by simple protonation by the photo-excited matrix, leading to the formation of the typical [M+X]+ type species (where X= H, Li, Na, K, etc.). Some multiply charged species, dimers and trimers can also be formed. Negative ions are formed from reactions involving deprotonation of the analyte by the matrix to form [M-H]- and from interactions with photoelectrons to form the [M]-° radical molecular ions.

These ionization reactions occur in the first tens of nanoseconds after irradiance, and within the initial desorbing matrix/analyte cloud. It is in this way that the characteristic MALDI spectra are created, typically giving large signals for species of the type [NM+X]n+ (N * n).

slide50

MALDI. Formation of ions by laser desorption

http://masspec.scripps.edu/information/intro/chapter3.html#3.2.1.

slide51

Sequence of events in MALDI.

  • Dried mixture of analyte and matrix on sample probe inserted into backplate of ion source.
  • (b1) Enlarged view of laser pulse striking sample.
  • (b2) Matrix is ionized and vaporized by laser and transfers some charge to analyte.
  • (b3) Vapor expands in a supersonic plume
slide52

Peptides and glycopeptides

Peptides and proteins

Peptides, small proteins and oligonucleotides (<10 bases)

The key aspect of MALDI MS is to dilute and isolate macromolecules in a suitable matrix of highly laser light absorbing small organic molecules, such as a-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA) and 2,5-dihydroxybenzoic acid (DHB), and then allowing it to dry on a MALDI-target into a crystalline deposit throughout which the molecules of the analyte are dispersed.

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MALDI-MS Sample preparation

Materials

For peptide analysis:

20 mg/ ml a-cyano-4-hydroxy-trans-cinnamic acid in 0.1% TFA/ 50% acetonitrile or in acetone-water (99:1, v/v).

20 mg/ ml 2,5-dihydroxybenzoic acid (DHB) in 0.1% TFA/ 30%* acetonitrile.

*The percentage of acetonitrilemay be varied, depending on the hydrophobicity of the peptides analyzed. The matrix additive 2-methoxy-5-benzoic acid may be added (1 mg/ml) to the DHB matrix solution for better detection of larger (>3000 Da) peptides.

For protein analysis:

sinapinic acid (20 mg/ ml) in 0.1% TFA/ 50% acetonitrile or in acetone-water (99:1, v/v).

http://www.bio.vu.nl/mnb/proteomics/8.maldisampleprep.html

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Electrospray Ionization (ESI)

Theory

The production of ions by evaporation of charged droplets obtained through spraying or bubbling, has been known about for centuries, but it was only fairly recently discovered that these ions may hold more than one charge4. A model for ion formation in ESI, containing the commonly accepted themes, is described below5:

Large charged droplets are produced by 'pneumatic nebulization'; i.e. the forcing of the analyte solution through a needle (see figure), at the end of which is applied a potential - the potential used is sufficiently high to disperse the emerging solution into a very fine spray of charged droplets all at the same polarity. The solvent evaporates away, shrinking the droplet size and increasing the charge concentration at the droplet's surface. Eventually, at the Rayleigh limit, Coulombic repulsion overcomes the droplet's surface tension and the droplet explodes. This 'Coulombic explosion' forms a series of smaller, lower charged droplets. The process of shrinking followed by explosion is repeated until individually charged 'naked' analyte ions are formed. The charges are statistically distributed amongst the analyte's available charge sites, leading to the possible formation of multiply charged ions under the correct conditions. Increasing the rate of solvent evaporation, by introducing a drying gas flow counter current to the sprayed ions (see figure), increases the extent of multiple-charging. Decreasing the capillary diameter and lowering the analyte solution flow rate i.e. in nanospray ionization, will create ions with higher m/z ratios (i.e. it is a softer ionization technique) than those produced by 'conventional' ESI and are of much more use in the field of bioanalysis.

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Gas-phase ion formation.

Electrospray interface for capillary electrophoresis / mass spectrometry.

Electrospray from a silica capillary.

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Atmospheric Pressure Chemical Ionization (APCI)

Similar to electrospray ionization, liquid effluent is introduced directly into the Atmospheric Pressure Chemical Ionization (APCI) source, however the similarity with electrospray stops there. The APCI source contains a heated vaporizer which facilitates rapid desolvation/vaporization of the droplets. Vaporized sample molecules are carried through an ion-molecule reaction region at atmospheric pressure. The ionization occurs through a corona discharge, creating reagent ions from the solvent vapor. Chemical ionization of sample molecules is very efficient at atmospheric pressure due to the high collision frequency. Proton transfer (protonation MH+ reactions) occurs in the positive mode, and either electron transfer or proton transfer (proton loss, [M-H]-) in the negative mode. The moderating influence of the solvent clusters on the reagent ions, and of the high gas pressure, reduces fragmentation during ionization and results in primarily molecular ions. APCI is widely used in the pharmaceutical industry to analyze relatively nonpolar, semi volatile samples of less than 1200 Daltons and it is an especially good ionization source for liquid chromatography.

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Atmospheric pressure chemical ionization interface between a liquid chromatography column and a mass spectrometer.

A fine aerosol is produced by the nebulizing gas flow and the heater. The electric discharge from the corona needle creates gaseous ions from the analyte.

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Mass analyzer: A device that separates a mixture of ions by their mass- to- charge ratios.

mass-to-charge ratio m/z:

The abbreviation m/ z is used to denote the dimensionless quantity formed by dividing the mass number of an ion by its charge number. It has long been called the mass- to- charge ratio although m is not the ionic mass nor is z a multiple or the elementary (electronic) charge e. The abbreviation m/z therefore, is not recommended. [IUPAC Compendium] 

mass analysis:

A process by which a mixture of ionic or neutral species is identified according to the mass- to- charge (m/ z) ratios (ions) or their aggregate atomic masses (neutrals). The analysis may be qualitative and/ or quantitative. [IUPAC Compendium]

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Quadrupole Mass Spectrometer

Introduction

A quadrupole mass filter consists of four parallel metal rods arranged as in the figure below. Two opposite rods have an applied potential of (U+Vcos(wt)) and the other two rods have a potential of -(U+Vcos(wt)), where U is a dc voltage and Vcos(wt) is an ac voltage. The applied voltages affect the trajectory of ions traveling down the flight path centered between the four rods. For given dc and ac voltages, only ions of a certain mass-to-charge ratio pass through the quadrupole filter and all other ions are thrown out of their original path. A mass spectrum is obtained by monitoring the ions passing through the quadrupole filter as the voltages on the rods are varied. There are two methods: varying w and holding U and V constant, or varying U and V (U/V) fixed for a constant w.

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Instrumentation

Quadrupole mass spectrometers consist of an ion source, ion optics to accelerate and focus the ions through an aperture into the quadrupole filter, the quadrupole filter itself with control voltage supplies, an exit aperture, an ion detector and electronics, and a high-vacuum system.

Quadrupole mass spectrometer.

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Magnetic-sector mass spectrometer

The ion optics in the ion-source chamber of a mass spectrometer extract and accelerate ions to a kinetic energy given by:

K.E. = 0.5 mv2 = eV

where m is the mass of the ion, v is it's velocity, e is the charge of the ion and V is the applied voltage of the ion optics.

The ions enter the flight tube between the poles of a magnet and are deflected by the magnetic field, H. Only ions of mass-to-charge ratio that have equal centrifugal and centripetal forces pass through the flight tube:

mv2 / r = Hev

centrifugal = centripetal forces.

Where r is the radius of curvature of the ion path:

r= mv / eH

This equation shows that the m/e of the ions that reach the detector can be varied by changing either H or V.

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Instrumentation

Single Focusing analyzers:

A circular beam path of 180, 90, or 60 degrees can be used. The various forces influencing the particle separate ions with different mass-to-charge ratios.

Double Focusing analyzers:

An electrostatic analyzer is added in this type of instrument to separate particles with difference in kinetic energies.

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Electrostatic sector of a double-focusing mass spectrometer. Positive ions are attracted toward the negative cylindrical plate. Trajectories of high-energy ions are changed less than trajectories of low-energy ions. Ions reaching the exit slit have a narrow range of kinetic energies.

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Ion-Trap Mass Spectrometry

Introduction

The ion-trap mass spectrometer uses three electrodes to trap ions in a small volume. The mass analyzer consists of a ring electrode separating two hemispherical electrodes. A mass spectrum is obtained by changing the electrode voltages to eject the ions from the trap. The advantages of the ion-trap mass spectrometer include compact size, and the ability to trap and accumulate ions to increase the signal-to-noise ratio of a measurement.

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Ion Trap Analysis 

Theory

The quadrupole ion trap mass analyser (see figure) consists of three hyperbolic electrodes: the ring electrode, the entrance endcap electrode and the exit endcap electrode. These electrodes form a cavity in which it is possible to trap and analyse ions. Both endcap electrodes have a small hole in their centres through which the ions can travel. The ring electrode is located halfway between the two endcap electrodes.

Ions produced from the source enter the trap through the inlet focussing system and the entrance endcap electrode. Various voltages are applied to the electrodes to trap and eject ions according to their mass-to-charge ratios. The ring electrode RF potential, an a.c. potential of constant frequency and variable amplitude, is applied to the ring electrode to produce a 3D quadrupolar potential field within the trapping cavity. This will trap ions in a stable oscillating trajectory confined within the trapping cell. The nature of the trajectory is dependent on the trapping potential and the mass-to-charge ratio of the ions. During detection, the electrode system potentials are altered to produce instabilities in the ion trajectories and thus eject the ions in the axial direction. The ions are ejected in order of increasing mass-to-charge ratio, focussed by the exit lens and detected by the ion detector system.

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Ion-trap mass spectrometer.

(a) Mass analyzer consists of two end caps (left and right) and central ring electrode. (b) Schematic diagram.

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Time-of-Flight Mass Spectrometry (TOF-MS)

Introduction

A time-of-flight mass spectrometer uses the differences in transit time through a drift region to separate ions of different masses. It operates in a pulsed mode so ions must be produced or extracted in pulses. An electric field accelerates all ions into a field-free drift region with a kinetic energy of qV, where q is the ion charge and V is the applied voltage. Since the ion kinetic energy is 0.5mv2, lighter ions have a higher velocity than heavier ions and reach the detector at the end of the drift region sooner.

Theory

K.E. = qV

1/2 mv2 = qV

v = (2qV/m)1/2

The transit time (t) through the drift tube is L/V where L is the length of the drift tube.

t = L / (2V/m/q)1/2

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Instrumentation

This schematic shows ablation of ions from a solid sample with a pulsed laser. The reflectron is a series of rings or grids that act as an ion mirror. This mirror compensates for the spread in kinetic energies of the ions as they enter the drift region and improves the resolution of the instrument. The output of an ion detector is displayed on an oscilloscope as a function of time to produce the mass spectrum.

Schematic of a reflectron TOF-MS

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Fourier Transform Ion Cyclotron Resonance (FT-ICR)

Theory

Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry is probably the most complex method of mass analysis. The technique has gone through a very complex development since it's conception in the mid. 1950s, this is reviewed in the history section. It is the most sensitive of the techniques in common use today, and has almost unlimited mass resolution, >106 is observable with most instruments and resolutions in the 104 to 105 range are routinely available.

The FT-ICR mass spectrometer consists of three main sections. The first is the sample source, which can be practically any of the available techniques, although ESI and MALDI are the most common. The second section is the ion transfer region, where the ions produce in the source are focussed, bunched and transferred into the high vacuum region before entering the analyser cell. The final region is the cell itself. It is possible in some cases (especially with MALDI) to produce ions directly in the cell, this does away with the need for the complex ion focussing and pumping regions.

The standard arrangement for the analyser region, of the FT-ICR instrument, is an ion-trap located within a spatially uniform static magnetic field of strength, B0. The effect of the magnetic field is to constrain the incident ions in a circular orbit (see figure), the frequency of which is determined by the mass, mi, charge, zi, and velocity, v, of the ion, by action of the Lorentz force, defined in equation 1. The analyte ion is bent into a circular path in a plane perpendicular to the magnetic field. The ion's angular frequency, wc, is defined by equation 2. The opposite sense of rotation is experienced by ions of opposite charge.

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Equation 1:

  • Equation 2:
  • Equation 3:
        • Where:
          • F = Lorentz force (observed by the incident ion)
          • B0 = ICR magnetic field
          • mi = mass of the ion
          • zi = charge on the ion
          • v = incident velocity of the ion
          • wc = induced rotational (cyclotron) frequency
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The presence of ions between a pair of detector electrodes (in the trapping cell) will not actually produce any measurable signal. It is necessary to excite the ions of a given m/z as a coherent package to a larger orbital radius, by applying an RF sweep of a few milliseconds across the cell (see figure). One frequency will excite one particular m/z (Fourier transformation allows for all frequencies to measure simultaneously). Measurement of the angular frequency (equation 2) leads to values for m/z (equation 3) and thus to the mass spectrum. Because frequency can be measured more accurately than any other physical property, the technique has a very high mass resolution. After excitation, the ions are allowed to relax back to their natural ICR motion for later remeasurement, if required.

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A schematic diagram of the trapping, excitation and detection of a ions, to produce a mass spectrum, in FT-ICR mass spectrometry:

http://www-methods.ch.cam.ac.uk/meth/ms/theory/fticr.html

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Ion Detection

Once the ion passes through the mass analyzer it is then detected by the ion detector, the final element of the mass spectrometer. The detector allows a mass spectrometer to generate a signal current from incident ions by generating secondary electrons, which are further amplified. Alternatively, some detectors operate by inducing a current generated by a moving charge. Among the detectors described, the electron multiplier and scintillation counter are the most commonly used and convert the kinetic energy of incident ions into a cascade of secondary electrons

Figure. After an ion passes through the Mass Analyzer a signal is produced by the detector.

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Ion Detectors

- Faraday cup

- Electron Multiplier

- High-energy dynodes with electron multiplier

- Array

- FT-MS

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Tandem Mass Spectrometry

In contrast to electron ionization (EI) which produces many fragment ions, the new ionization techniques are relatively gentle and do not produce a significant amount of fragment ions. To obtain more information on the molecular ions generated in the electrospray ionization and MALDI ionization sources, it has been necessary to apply techniques such as tandem mass spectrometry (MS/MS) to induce fragmentation. Tandem mass spectrometry (abbreviated MSn - where n refers to the number of generations of fragment ions being analyzed) allows one to induce fragmentation and mass analyze the fragment ions. This is accomplished by collisionally generating fragments from a selected ion and then mass analyzing the fragment ions. Fragmentation can be achieved by inducing ion/molecule collisions by a process known as collision-induced dissociation (CID) (also known as collision-activated dissociation (CAD)). Collision-induced dissociation is accomplished by selecting an ion of interest with a mass analyzer and introducing that ion into a collision cell. The selected ion then collides with a collision gas (typically argon or helium) resulting in fragmentation. The fragments are then analyzed to obtain a fragment ion spectrum. The abbreviation MSn is applied to processes which analyze beyond the initial ions (MS) to the fragment ions (MS2) and subsequent generations of fragment ions (MS3, MS4 and …). Tandem mass analysis is primarily used to obtain structural information.

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Multiple Mass Spectrometry ; MS/MS/MS:

Provides even greater certainty of identification and additional characterization information than electrospray ionization/ tandem mass spectrometry. Fully automated. [CHI Proteomics report] 

When more than two stages are involved, the technique is called multi- dimensional MS (MS n where n indicates the number of stages).

Principle of selected reaction monitoring, also called tandem mass spectrometry, mass spectrometry / mass spectrometry, or MS/MS

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Fragments are analyzed to obtain a fragment ion spectrum. The abbreviation MSn is applied to processes which analyze beyond the initial ions (MS) to the fragment ions (MS2) and subsequent generations of fragment ions (MS3, MS4 and …).

http://masspec.scripps.edu/information/intro/chapter3.html#3.2.1.