mass spectrometry? - personal webpages at ntnufolk.ntnu.no/audunfor/7. semester/spektro vk/ms...3 5...

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1

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

4

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

?


6

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).

8

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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9

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

10

Ionization Methods


Solid-state:




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11

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

12

CH3

CH3

CH3CH3

CH3CH3

CH3

Mass=170Mass=170

NB! Due to fragmentation yopu will not alwaysfind your molecular ion

Exampel:

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13

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

14

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


16

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


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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

18

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


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19

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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21

Ionization Methods


Solid-state:




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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.


24

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


26

Solvent requirements :

Volatile solvents required – b.p. < 100°C (H2O).

MALDI ionization

14

27

OR

+

OR

Sample

+

Matrix

+

OR

4 × 15 seconds

MALDI ionization: Sample preparation-Solvent-free preparation


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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

14

EI best; e- capture may be possible in negative mode under appropriate conditions

13

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.

1

CommentsMALDI-MALDI+CI-CI+EI

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Ionization Methods


Solid-state:


Liquid-phaseElectrospray (ESI)Atmosferic pressure chemical inization (APCI) Atmosferic pressure photoionization (APPI )ASAP


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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


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ESI – Multiple Charging

~16,700 Daltons


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


44Solvent Effects

[M+MeOH+H]+

[M+H]+

[M+MeOH+Na]+

[M+H]+

[2M+H]+


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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


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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

48

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

50

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


Solid-state:




54

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

56

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

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Ionization Methods: Combined with Chromatography


mass spectrometry? - personal webpages at ntnufolk.ntnu.no/audunfor/7. semester/spektro vk/ms...3 5...

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