mass spectrometry? - personal webpages at ntnufolk.ntnu.no/audunfor/7. semester/spektro vk/ms...3 5...
TRANSCRIPT
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MS program
20.08.2013MS: Ionization methods
22.08.2013MS: Ionization methods + Analytical Information
27.08.2013MS: Analytical Information + Analyzers
29.08.2013MS: Analyzers + Questions
03.09.2013MS: Lab time
Compendium Sections which are not required:1.2.5;1.6;1.7;1.8.3; 1.8.4; 1.9; 1.11.5; 1.15; 1.16 4.2.11; 4.3.7; 4.7.9
KJ3022 MS compendium gives a deeper explanation of what is mentioned in the slides
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Mass Spectrometry?
(mainlib) Benzoic Acid20 30 40 50 60 70 80 90 100 110 120 130
0
50
100
2739
45
51
6574
77
94
105122
HO O
EI +
m/z
% R
elat
ive
Abu
ndan
ce
2
3
(mainlib) Benzoic Acid20 30 40 50 60 70 80 90 100 110 120 130
0
50
100
2739
45
51
6574
77
94
105122
HO O
m/z
% R
elat
ive
Abu
ndan
ce
MS information:
•Mass (m/z) – nominal or accurate (molecular ion)
•Isotopic pattern
• Fragmentation
122-17
ATOMIC COMPOSITION AND STRUCTURE
Mass/charge ratio
EI + ionization mode
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Data collection =Detector
Mass spectrometry prosess
Ion acceleration and separation = Analyzer
Ionization = ion source
Courtesy of EPSRC National Mass Spectrometry Service Center
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5
Data analysis
?
Courtesy of EPSRC National Mass Spectrometry Service Center
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CONCEPTS
Nominal mass is defined as the integer mass of the most abundant naturally occurring stable isotope of an element. The nominal mass of an element is often equal to the integer mass of the lowest mass isotope of that element, e.g., for H, C, N, O, S, Si, P, F, Cl, Br, I. The nominal mass of an ion is the sum of the nominal masses of the elements in its empirical formula.
The isotopic mass is the exact mass of an isotope. It is very close to but not equal to the nominal mass of the isotope.
Metastable ion:fragment ions, undergo secondary fragmentations in the analyzer tube of the mass spectrometer; the resulting “signals”or peaks represent neither the m/z of the first ion nor that of the second ion; instead, “metastable ion” peaks are observed
For a reactionF1
+→ F2+ a “metastable ion” peak, m*, is observed m* = m2
2/m1
(m/z)1 (m/z)2
metastable ion peaks require a special type of spectrometer; they give valuableinformation about fragmentation patterns of molecular ions.
Molecular ion of CO, nominal mass? 28 isotopic mass ? 27.9949
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7
____________________ nominal isotopic
carbon monoxide CO 28 27.9949molecular nitrogen N2 28 28.0061ethylene CH2=CH2 28 28.0313
Gln C5H10N2O3 146 146.0691Lys C6H4N2O2 146 146.1055
CONCEPTS :
Mass defect = difference between exact mass and integer mass of a nuclide*(*characterised by the no. of protons and neutrons in nucleus).
H atom (proton plus electron) 1.007825 Neutron 1.0086665
________predicted mass of D : 2.016490 Actual mass D 2.01410
The “missing mass” is the mass defect (it is explained by Einstein’s theory of mass-energy equivalence, E = mc2, and represents the energy required to bind the atomic nucleus together).
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Ionization Methods
Gas-phaseElectron Impact (EI)Chemical Ionization (CI)Field ionization (FI)
Solid-state:
Field desorptionMALDI (matrix-assisted laser desorption/ionization)SIMS (Secondary ion mass spectrometry)Plasma desorptionFast atom bombardement (involatile liquid matrix)
Liquid-phaseElectrospray (ESI)Atmosferic pressure chemical inization (APCI) Atmosferic pressure photoionization (APPI )
Direct analysis in real time ( DART )Desorption electrospray ionization (DESI)
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Ionisation method: WHY SO MANY!! ?
N
S
PtS
Pt
S
O
O
N
S
N
O
O
O
O
OHO
OHOH
O
OHO
OH
O
HO
O
OH
HO O
OH
HO
HN
O
O
MANY DIFFERENT
MOLECULES
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Ionization Methods
Gas-phaseElectron Impact (EI)Chemical Ionization (CI)Field ionization (FI)
Solid-state:
Field desorptionMALDI (matrix-assisted laser desorption/ionization)SIMS (Secondary ion mass spectrometry)Plasma desorptionFast atom bombardement (involatile liquid matrix)
Liquid-phaseElectrospray (ESI)Atmosferic pressure chemical inization (APCI) Atmosferic pressure photoionization (APPI )
Direct analysis in real time ( DART )Desorption electrospray ionization (DESI)
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Ionization method: Electron Impact EI
Gas phase molecules are irradiated by beam of electrons
Electron ejection M + eM + e-- MM++•• + 2e+ 2e--
molecular ion
Fragments
thermostable
Molecular ion
Heated filament ofrhenium or tungsten
Fragment only positive ions
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CH3
CH3
CH3CH3
CH3CH3
CH3
Mass=170Mass=170
NB! Due to fragmentation yopu will not alwaysfind your molecular ion
Exampel:
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Advantages :
Stabile and reliable methodRelatively high sensitivityCharacteristic spectra produced (Libraries like NIST )Indispensable tool for analysis of many small synthetic and naturally-occurring compounds
Disadvantages:
Unsuitable for poorly volatilised, thermally labile molecules and Ionic functional groups (salts etc.)Non-ionic groups involved in H-bondingMolecular weight often limited to < 1000Da; cleavage rather than volatilisation on heatingDuring EI, 1020eV energy is transferred to molecule, often leading to fragmentation …..M+. low or absent.
Ionisation method: Electron Impact EI
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Mild ionisation method
Reagent ions produced by EI of reagent gas (most commonly methane, isobutane or ammonia) at high pressure (1 x 10-4mbar).
For methane
For ammonia:
NH3 – e- NH3+. (unstable) + 2e-
NH3+. + NH3 NH4
+ + NH2.
These ions are only slightly reactive with reagent gas itself, but readily react to ionise the sample via ion-molecule reactions in which the reagent ions act as Brnsted acids (proton donors)
Ionization method : Chemical ionization CI
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4 categories of ion-molecule reactions
(1) Proton transfer: M + BH+ MH+ + B
(2) Charge exchange: M + X+. M+. + X
(3) Electrophilic addition: M + X+ MX+
(4) Anion Abstraction: AB + X+ B+ + AX
In the case of ammonia reagent gas, NH4+ can act as proton transfer (M+H)+
or enter into addition reaction (M+NH4)+
Relatively simple spectra = less fragmentation =Molecular ion can be observed
Ionization method : Chemical ionization CI
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205399 MW=243?CI+(NH3)QUATTRO
07-Jun-2004EPSRC National Centre Swansea
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290m/z0
100
%
52.2
46.339.3
260.1
182.1
90.158.2 63.278.2 104.1 121.1 169.0152.1136.1
184.0
242.0197.1203.1 213.1
228.1247.1
263.1
264.1296.0277.1
Ammonia CI spectrum showing (M+NH4)+
(M+NH4)+ ion @ m/z 260/262
(Br isotopes)
Ion =242 + 18
Courtesy of EPSRC National Mass Spectrometry Service Center
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Example: EI + CI Methane as reagent
Which is the molecular ion ? MW=208What has happened with methane and our molecule? [M+H]+=209 and [M+C2H5]
+=237Any other obvious information from Isotopes and fragmentation?
EI
[M+H]+
[M+C2H5]+
-18
HO Br
O
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Advantages :
Mild Ionization Molecular weight determination possible via adduct ion formation.Positive and negative (electron capture) CI possibleMany sources are EI/CI combined sources Rules governing fragmentation CI complementary to EIManipulation of different reagent gases to influence structural information yielded
Disadvantages:
Gas phase technique: sample needs to be vaporisedLimits use of CI for high molecular weight molecules Analysis of organo-metallic and silylation compounds can lead to contamination of CI source
Ionization method : Chemical ionization CI
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Ionisation method: CI and EI probes
direct exposure probe (DEP)direct insertion probe (DIP)
DEP+CI = desorption chemical ionization (DCI).
In DCI, the analyte is applied from solution or suspension to the outside of a thinresistively heated wire loop or coil. Then, the analyte is directly exposed to the reagent gas plasma while being rapidly heated at rates of several hundred degrees per second .The rapid heating of the sample plays an important role in promoting molecularspecies rather than pyrolysis products.
reservoir inlets and gas chromatographs
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Atoms or molecules are ionized by action of a strong electric field (ionization by quantum mechanical tunneling of electrons) independent of the sample providing.
sample is supplied from a separate inlet system in the gaseous state,
FI produces M*+ with little or no fragmentationFI is also used for performing isotope ratio measurements on samples that either givesmall molecular ions or large (M-H)+ ions in EI.
3 differences between CI and FI:-less fragmentation in FI,-no high-resolution FI, and FI is less sensitive.
Ionisation method: Field ionization FI
10-micron diameter tungsten emitter wires with carbon whiskers
http://en.wikipedia.org/wiki/Field_desorption
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Ionization Methods
Gas-phaseElectron Impact (EI)Chemical Ionization (CI)Field ionization (FI)
Solid-state:
Field desorptionMALDI (matrix-assisted laser desorption/ionization)SIMS (Secondary ion mass spectrometry)Plasma desorptionFast atom bombardement (involatile liquid matrix)
Liquid-phaseElectrospray (ESI)Atmosferic pressure chemical inization (APCI) Atmosferic pressure photoionization (APPI )
Direct analysis in real time ( DART )Desorption electrospray ionization (DESI)
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Ionisation methods: Solid-state: Desorption methods
desorption - changing from an adsorbed state on a surface to a gaseous or liquid state
FD (Field desorpotion) -Same principle as Field Ionization, but no needfor evaporation of the analyte. Good for non-polar molecules
Plasma desorption -not in used, substituted by MALDI
MALDI (matrix-assisted laser desorption/ionization)
SIMS (Secondary ion mass spectrometry)
Fast atom bombardement (involatile liquid matrix)
Use for : non-volatile high molecular mass and or thermally label molecules(polymers, proteins….etc)
Types:
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irradiation
H+
-
+desorption
desolvation
gas-phase ionisation
= Analyte molecule
= Matrix molecule
MALDI ionization
Analyte embedded within a solid or liquid matrix.Matrix required to have strong absorption at the laser wavelength ( = 337 nm for typical N2 UV-laser).Irradiation induces rapid heating of the matrix, resulting in localised matrix sublimation into the gas phase.Intact analyte is simultaneously desorbed and ionised in the expanding matrix plume.
Courtesy of EPSRC National Mass Spectrometry Service Center
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MALDI has different lasers and matrix
*DCTB = trans-2-[3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile**DAC = Di-ammonium citrate
alpha -Cyano-4-hydroxycinnamic acid (-CHCA) Peptides/proteins (mass <10 kDa), carbohydratesSinapinic acid (SA) Peptides/proteins (mass >10 kDa), dendrimers2,5-Dihydroxybenzoic acid (DHB) Polar synthetic polymers, carbohydrates, organics1,8,9-Trihydroxyanthracene or Dithranol (Dith) Synthetic polymers, dendrimers, organics2,4,6-Trihydroxyacetophenone (THAP) +DAC** Oligonucleotides** (mass <3.5 kDa), acidic carbohydrates3-Hydroxypicolinic acid (HPA) +DAC** Oligonucleotides** (mass >3.5 kDa)2'-(4-Hydroxyphenylazo)benzoic acid (HABA) Cyclic peptides, synthetic polymersDCTB* Inorganics, organometallics, fullerenestrans -3-Indole acrylic acid (IAA) Non-polar synthetic polymers7,7,8,8-Tetracyanoquinodimethane (TCNQ) Polyaromatic hydrocarbons (PAHs)Succinic acid (IR laser) Peptides/proteins, synthetic polymers
Matrix Application
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MALDI ionization : Sample preparation -Dried-droplet preparation
AnalyteMatrix
Analyte and matrix mixed
Sample
Sample deposition onto slide
Solvent evaporation
irradiationMatrix stronglyabsorbs at thelaser wavelength
Excitation
Courtesy of EPSRC National Mass Spectrometry Service Center
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Solvent requirements :
Volatile solvents required – b.p. < 100°C (H2O).
MALDI ionization
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OR
+
OR
Sample
+
Matrix
+
OR
4 × 15 seconds
MALDI ionization: Sample preparation-Solvent-free preparation
Courtesy of EPSRC National Mass Spectrometry Service Center
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Advantages
Mild ionisation of a wide variety of analytes.
High sensitivity - 5 fmol.
Relatively high tolerance to salt and buffer impurities.
Very wide mass range covered. theoretically almost unlimited; in practice, limits can be as low as 3000 u, e.g., with polyethylene, or as high as 300,000 u in case of antibodies.
Generally, only singly charged ions observed.
Disadvantages:
Sample preparation can be problematic/“dark art” and slow. Automation not practical for chemically different analytes.
Significant chemical background from matrix, lowest mass range can be sometimes as low as 1000m/z
Relatively large amount of sample preferred for ease of handling, and required for polymeric or solvent-free preparations.
MALDI ionization
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MALDI ionization: Application example
http://edge.rit.edu/content/P08043/public/Home
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Secundary ions emitted are analysedafter irradiation of the surface with a energetic primary ions beam
SIMS: secondary ion mass spectrometry (organic and inorganic conducting-surface analysis)
Static source- no damage on surfaceSIMS Low current primary ion beam)
Dynamic source-surface erosion
LSIMS uses Cs+ ionssample disolved in non volatile matrix, such as glycerol ……
http://www.ifw-dresden.de/institutes/institute-for-complex-materials/departments/micro-and-nanostructures/available-methods/secondary-ion-mass-spectrometry-sims/
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FAB: fast atom bombardment (organic applications)
Liquid matrix as employed in FAB and LSIMS= non volatileIt absorbs the primary energy. By solvation it helps to overcome intermolecular forces between analyte molecules or ions. It provides a continuously refreshing and long-lasting supply of analyte.
Ion formation: proton donating/accepting or electron donating/accepting species upon bombardment
the surface is bombarded chiefly by neutral atoms/molecules of argon or preferably xenon
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SIMS: Application example
Tablet Cross Section
The example below shows mass resolved secondary ion images from a tablet cross section.
This technique can be used to determine the distribution of the differentingredients, including the drug itself, within the tablet.
http://www.ion-tof.com/applications-pharmaceuticals-IONTOF-TOF-SIMS-TIME-OF-FLIGHT-SURFACE-ANALYSIS.htm
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O
Cl
O
OH
O
O
SiMe3
OCl
OHO
1
2
3
4
5
13
14
15
ESI+ with NH4OAc (or similar) added yyy15
Ammonia CI bad with halogens; whatever mode is used to avoid use of methanol! (will react with Cl
here ?y14
EI best; e- capture may be possible in negative mode under appropriate conditions
?:)13
CI with methane OK, ammonia would not work yf5
CI with methane OK, ammonia would not work?:)4
CI with ammonia good; EI probably OK if molecule doesn’t fragment; ESI may struggle with such low
MW.?:)y3
yyf2
EI will work but probably not show M+ ([M-57] = most likely - why?); pos CI: hope for NH4+ addition at O; CI neg: hope for electron capture at halogen; ESI+ would need NH4+ (or similar) added to assist
ionisation.
?yf1
CommentsMALDI-MALDI+CI-CI+EI
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O
Cl
O
OH
O
O
SiMe3
OCl
OHO
1
2
3
4
5
13
14
15
ESI+ with NH4OAc (or similar) added 15
Ammonia CI bad with halogens; whatever mode is used to avoid use of methanol! (will react with Cl here
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EI best; e- capture may be possible in negative mode under appropriate conditions
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CI with methane OK, ammonia would not work 5
CI with methane OK, ammonia would not work4
CI with ammonia good; EI probably OK if molecule doesn’t fragment; ESI may struggle with such low
MW.3
2
EI will work but probably not show M+ ([M-57] = most likely - why?); pos CI: hope for NH4+ addition at O; CI neg: hope for electron capture at halogen; ESI+
would need NH4+ (or similar) added to assist ionisation.
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CommentsMALDI-MALDI+CI-CI+EI
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Ionization Methods
Gas-phaseElectron Impact (EI)Chemical Ionization (CI)Field ionization (FI)
Solid-state:
Field desorptionMALDI (matrix-assisted laser desorption/ionization)SIMS (Secondary ion mass spectrometry)Plasma desorptionFast atom bombardement (involatile liquid matrix)
Liquid-phaseElectrospray (ESI)Atmosferic pressure chemical inization (APCI) Atmosferic pressure photoionization (APPI )ASAP
Direct analysis in real time ( DART )Desorption electrospray ionization (DESI)
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Your turn to teach !
Make 4 groups and try in the next 20 minutes to find information about one of the 4 different techniques.
Group 1 ESI Group 2 APPIGroup 3 APCIGroup 4 ASAP
-what type of ion is being formed ? -Why is this technique so important ? Which type of molecules can be analyzed ?
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Ionization methods- Liquid phase : Atmospheric pressure ionization
Soft ionizationMass spectra provide mainly molecular weight information.Highly efficient production of ions.Different types : ESI, APCI and APPI
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Ionisation method : Electrospray ESI
NO fragmentation, but possible side reactions Polar molecules or molecules with heteroatomes
Ions are generated by ion transfer : (M+H)+, (M+Na)+, (M+NH4)+
Multiply charged ions (M+2H)2+ or (M+10H)10+
Possible Dimer/trimer formation
Good for Organometallic salts
OH
OHHH
M MH+H
http://www.rsc.org/chemistryworld/Issues/2003/February/together.asp
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Ionisation method : Electrospray ESI
http://www.chm.bris.ac.uk/ms/theory/esi-ionisation.html
http://www.youtube.com/watch?v=paIKIu1-ChA
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ESI Ion Formation
Very high mass range >200,000 (m/z reduced due to multiple charging, z increases).
Low salt and buffer tolerance, these will compete with the analyte during ionisation. (Ion suppression )
But can also enhance ion formation. NH4OAc often promotes [M+NH4]+ (positive mode)
? Multiple charging can be confusing.
Modification of sample pH can induce ionisation and increase sensitivity or promote multiple charging.
Sensitivity is Concentration dependant
Positive Mode:
[M+H]+, [M+nH]n+, [M+NH4]+, [M+Na]+, [M+K]+, [2M+H]+, [2M+Na]+, etc…..
Negative Mode:
[M-H]-, [M-nH]n-, [M+Cl]- etc…..
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ESI – Singly Charged
[M+H]+
[M+NH4]+
[M+Na]+
M/W = 281
Courtesy of EPSRC National Mass Spectrometry Service Center
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ESI – Multiple Charging
~16,700 Daltons
Courtesy of EPSRC National Mass Spectrometry Service Center
Myoglobin : oxygen transporting protein
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Mass spectrum of Myoglobin, observed in the form [M+nH]n+.
500 800 1100 1400 1700 2000m/z
14+
12+
10+
16+
18+20+
22+
24+
21+ ~808 Da21*808=16968 Da(this carries +21H)
16968-21 = 16947Da
21+
ESI – Multiple Charging
Courtesy of EPSRC National Mass Spectrometry Service Center
44Solvent Effects
[M+MeOH+H]+
[M+H]+
[M+MeOH+Na]+
[M+H]+
[2M+H]+
Courtesy of EPSRC National Mass Spectrometry Service Center
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Information from Electrospray
No Molecular ion (M+H)+
Onlyammonium adduct (M+NH4)+
MW : 501 + MW NH4: 18
Ion found : 519 ammonium adduct
MW : 501
Courtesy of EPSRC National Mass Spectrometry Service Center
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Ions formed [M+H]+ or [M-H]-
APCI not multiple charged ions of the type [M+nH]n+
Tolerates variety of solvents and buffers.
Analyte must be volatile and thermally stable.
Ions are formed in the gas phase similar to the ionization process of CI.
Normal phase solvent
High flow rates
Interface to HPLC
Thermal stabilityInsensitive to salts
Volatile sampleMolecular species
DisadvantagesAdvantages
APCI : Atmospheric pressure chemical ionization
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Photon source photoionize vapor molecules upon exit from the vaporizer
Requires right combination of uv lamp , solvent, analyte and sometime dopant (for example toluene)
Rule of thumb, recommended for compounds with a UV active moiety
APPI : Atmospheric pressure photoionization
Positive ions [M+H]+, M*+
http://www.chem.agilent.com/en-US/products-services/Instruments-Systems/Mass-Spectrometry/Atmospheric-Pressure-Photoionization-Source-(APPI)/Pages/gp2294.aspx
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Courtesy of Agilent
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ASAP technique (Waters™)
Ion generation in positive-jon mode comes about by corona discharge, forming both radical cations (M+.) and protonated cations (M+H)+.
Ionization technique : proton transfer or charge transfer (dry conditions without lock spray or moist)
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ElementElement Mass % Mass % Mass % TypeH 1 100 2 0.016 AC 12 100 13 1.08 A + 1
N 14 100 15 0.36 A + 1
O 16 100 17 0.04 18 0.20 A + 2F 19 100 ASi 28 100 29 5.1 30 3.4 A + 2P 31 100 AS 32 100 33 0.80 34 4.4 A + 2
Cl 35 100 37 32.5 A + 2
Br 79 100 81 98.0 A + 2I 127 100 A
A A + 1 A + 2
Natural Isotopes and their relative amount in nature
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8 9 10 11 12 13 14 150
100
%
12.0
C1
116 117 118 119 120 121 122 123 10
100
%
120.0
121.0
C10
Carbon isotopes - examples
C1C10
1196 1198 1200 1202 1204 12060
100
%
1201.01200.0
1202.0
1203.0
1204.0
C100
12000 12005 12010 12015 120200
100
%
12011.012010.012009.0
12008.0
12007.0
12006.0
12005.0
12012.0
12013.0
12014.0
12015.0
12016.1
12017.1
C1000C100C1000
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Ionization Methods
Gas-phaseElectron Impact (EI)Chemical Ionization (CI)Field ionization (FI)
Solid-state:
Field desorptionMALDI (matrix-assisted laser desorption/ionization)SIMS (Secondary ion mass spectrometry)Plasma desorptionFast atom bombardement (involatile liquid matrix)
Liquid-phaseElectrospray (ESI)Atmosferic pressure chemical inization (APCI) Atmosferic pressure photoionization (APPI )
Direct analysis in real time ( DART )Desorption electrospray ionization (DESI)
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DESI: Desorption electrospray ionization
ESI + Direct probe exposure = DESI
Main advantage is able to map compoundsposition on the native surface
Principles is :Solvent ionized by ESI is sprayed on the analyte surface
Mechanism is not yet established
www.medgadget.com/2006/10/desi_delivers_i.html
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DART: Direct analysis in real time Direct detection of chemicals on surface, in liquids or in gases withoutthe need of sample preparation
Penning ionization:transfer of energy from the excited gas to the analyte (positive and negative ions)
Proton transfer is also possible when using He as gas and due to water clusters
Non multiply charge ions are obtained with DART.
http://commons.wikimedia.org/wiki/File:DART_ion_source_capsule.jpg
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Ionization Methods: How to choose ?
Take a look at your molecule, info you need and type of sample
Molecule : -Size -Polarity -Thermo stability -Mixture of compounds
Information:-Structure elucidation -quantification -Formula confirmation
Type of sample :-Solid -Liquid -Gas