KJ3022 MS compendium gives a deeper explanation of what is mentioned in the slides

Transcription

1 1 MS program MS: Ionization methods MS: Ionization methods + Analytical Information MS: Analytical Information + Analyzers MS: Analyzers + Questions MS: Lab time KJ3022 MS compendium gives a deeper explanation of what is mentioned in the slides Compendium Sections which are not required: 1.2.5;1.6;1.7;1.8.3; 1.8.4; 1.9; ; 1.15; ; 4.3.7; Mass Spectrometry? % Relative Abundance H 50 EI (mainlib) Benzoic Acid m/z 122 1

2 3 % Relative Abundance EI ionization mode (mainlib) Benzoic Acid 77 m/z MS information: Mass (m/z) nominal or accurate (molecular ion) Isotopic ATMIC pattern CMPSITIN AND STRUCTURE Fragmentation H Mass/charge ratio Mass spectrometry prosess Ionization = ion source Ion acceleration and separation = Analyzer Data collection = Detector Courtesy of EPSRC National Mass Spectrometry Service Center 2

3 5 Data analysis? Courtesy of EPSRC National Mass Spectrometry Service Center 6 CNCEPTS 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,, 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 reaction F 1+ F + 2 a metastable ion peak, m*, is observed m* = m 22 /m 1 (m/z) 1 (m/z) 2 metastable ion peaks require a special type of spectrometer; they give valuable information about fragmentation patterns of molecular ions. Molecular ion of C, nominal mass? 28 isotopic mass?

4 7 CNCEPTS : nominal isotopic carbon monoxide C molecular nitrogen N ethylene CH 2 =CH Gln C 5 H 10 N Lys C 6 H 4 N 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) Neutron predicted mass of D : Actual mass D The missing mass is the mass defect (it is explained by Einstein s theory of massenergy equivalence, E = mc2, and represents the energy required to bind the atomic nucleus together). 8 Ionization Methods Gas-phase Solid-state: Liquid-phase Electron Impact (EI) Chemical Ionization (CI) Field ionization (FI) Field desorption MALDI (matrix-assisted laser desorption/ionization) SIMS (Secondary ion mass spectrometry) Plasma desorption Fast atom bombardement (involatile liquid matrix) Electrospray (ESI) Atmosferic pressure chemical inization (APCI) Atmosferic pressure photoionization (APPI ) Direct analysis in real time ( DART ) Desorption electrospray ionization (DESI) 4

5 9 Ionisation method: WHY S MANY!!? N S Pt Pt S S N S MANY DIFFERENT MLECULES N H H H H H H H H H H H N 10 Ionization Methods Gas-phase Solid-state: Liquid-phase Electron Impact (EI) Chemical Ionization (CI) Field ionization (FI) Field desorption MALDI (matrix-assisted laser desorption/ionization) SIMS (Secondary ion mass spectrometry) Plasma desorption Fast atom bombardement (involatile liquid matrix) Electrospray (ESI) Atmosferic pressure chemical inization (APCI) Atmosferic pressure photoionization (APPI ) Direct analysis in real time ( DART ) Desorption electrospray ionization (DESI) 5

6 11 Ionization method: Electron Impact EI thermostable Gas phase molecules are irradiated by beam of electrons Heated filament of rhenium or tungsten Electron ejection M + e - M + + 2e - molecular ion Fragment only positive ions Molecular ion Fragments 12 Exampel: H 3 C H 3 C C H 3 H 3 C CH 3 CH 3 H 3 C Mass=170 NB! Due to fragmentation yopu will not always find your molecular ion 6

7 13 Ionisation method: Electron Impact EI Advantages : Stabile and reliable method Relatively high sensitivity Characteristic 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-bonding Molecular weight often limited to < 1000Da; cleavage rather than volatilisation on heating During EI, 10 20eV energy is transferred to molecule, often leading to fragmentation..m +. low or absent. 14 Ionization method : Chemical ionization CI Mild ionisation method Reagent ions produced by EI of reagent gas (most commonly methane, isobutane or ammonia) at high pressure (1 x 10-4 mbar). For methane For ammonia: NH 3 e- NH +. 3 (unstable) + 2e- NH NH 3 NH NH 2 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 Br nsted acids (proton donors) 7

8 15 Ionization method : Chemical ionization CI 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, NH 4 + can act as proton transfer (M+H) + or enter into addition reaction (M+NH 4 ) + Relatively simple spectra = less fragmentation =Molecular ion can be observed 16 Ammonia CI spectrum showing (M+NH 4 ) MW=243? EPSRC National Centre Swansea QUATTR Jun-2004 CI+(NH3) Ion = (M+NH 4 ) + m/z 260/262 (Br isotopes) % m/z Courtesy of EPSRC National Mass Spectrometry Service Center 8

9 17 Example: EI + CI Methane as reagent EI Which is the molecular ion? MW=208 What has happened with methane and our molecule? [M+H] + =209 and [M+C 2 H 5 ] + =237 Any other obvious information from Isotopes and fragmentation? [M+H] + H Br -18 [M+C 2 H 5 ] + 18 Ionization method : Chemical ionization CI Advantages : Mild Ionization Molecular weight determination possible via adduct ion formation. Positive and negative (electron capture) CI possible Many sources are EI/CI combined sources Rules governing fragmentation CI complementary to EI Manipulation of different reagent gases to influence structural information yielded Disadvantages: Gas phase technique: sample needs to be vaporised Limits use of CI for high molecular weight molecules Analysis of organo-metallic and silylation compounds can lead to contamination of CI source 9

10 19 Ionisation method: CI and EI probes direct insertion probe (DIP) direct exposure probe (DEP) reservoir inlets and gas chromatographs DEP+CI = desorption chemical ionization (DCI). In DCI, the analyte is applied from solution or suspension to the outside of a thin resistively 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 molecular species rather than pyrolysis products. 20 Ionisation method: Field ionization FI 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, 10-micron diameter tungsten emitter wires with carbon whiskers FI produces M* + with little or no fragmentation FI is also used for performing isotope ratio measurements on samples that either give small 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. 10

11 21 Ionization Methods Gas-phase Solid-state: Liquid-phase Electron Impact (EI) Chemical Ionization (CI) Field ionization (FI) Field desorption MALDI (matrix-assisted laser desorption/ionization) SIMS (Secondary ion mass spectrometry) Plasma desorption Fast atom bombardement (involatile liquid matrix) Electrospray (ESI) Atmosferic pressure chemical inization (APCI) Atmosferic pressure photoionization (APPI ) Direct analysis in real time ( DART ) Desorption electrospray ionization (DESI) 22 Ionisation methods: Solid-state: Desorption methods desorption - changing from an adsorbed state on a surface to a gaseous or liquid state Use for : non-volatile high molecular mass and or thermally label molecules (polymers, proteins.etc) Types: FD (Field desorpotion) Plasma desorption -Same principle as Field Ionization, but no need for evaporation of the analyte. Good for nonpolar molecules -not in used, substituted by MALDI MALDI (matrix-assisted laser desorption/ionization) SIMS (Secondary ion mass spectrometry) Fast atom bombardement (involatile liquid matrix) 11

12 23 MALDI ionization Analyte embedded within a solid or liquid matrix. Matrix required to have strong absorption at the laser wavelength ( = 337 nm for typical N 2 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. irradiation = Analyte molecule = Matrix molecule desolvation - desorption + H + gas-phase ionisation Courtesy of EPSRC National Mass Spectrometry Service Center 24 MALDI has different lasers and matrix Matrix Application alpha -Cyano-4-hydroxycinnamic acid ( -CHCA) Peptides/proteins (mass <10 kda), carbohydrates Sinapinic acid (SA) Peptides/proteins (mass >10 kda), dendrimers 2,5-Dihydroxybenzoic acid (DHB) Polar synthetic polymers, carbohydrates, organics 1,8,9-Trihydroxyanthracene or Dithranol (Dith) Synthetic polymers, dendrimers, organics 2,4,6-Trihydroxyacetophenone (THAP) +DAC** ligonucleotides** (mass <3.5 kda), acidic carbohydrates 3-Hydroxypicolinic acid (HPA) +DAC** ligonucleotides** (mass >3.5 kda) 2'-(4-Hydroxyphenylazo)benzoic acid (HABA) Cyclic peptides, synthetic polymers DCTB* Inorganics, organometallics, fullerenes trans-3-indole acrylic acid (IAA) Non-polar synthetic polymers 7,7,8,8-Tetracyanoquinodimethane (TCNQ) Polyaromatic hydrocarbons (PAHs) Succinic acid (IR laser) Peptides/proteins, synthetic polymers *DCTB = trans-2-[3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile **DAC = Di-ammonium citrate 12

13 25 MALDI ionization : Sample preparation -Dried-droplet preparation Analyte Matrix Sample Analyte and matrix mixed Sample deposition onto slide Matrix strongly irradiation absorbs at the laser wavelength Excitation Solvent evaporation Courtesy of EPSRC National Mass Spectrometry Service Center 26 MALDI ionization Solvent requirements : Volatile solvents required b.p. < 100 C (H 2 ). 13

14 27 MALDI ionization: Sample preparation-solvent-free preparation Sample + Matrix + R R + R 4 15 seconds Courtesy of EPSRC National Mass Spectrometry Service Center 28 MALDI ionization 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. 14

15 29 MALDI ionization: Application example 30 SIMS: secondary ion mass spectrometry (organic and inorganic conducting-surface analysis) Secundary ions emitted are analysed after irradiation of the surface with a energetic primary ions beam SIMS Static source- no damage on surface Low current primary ion beam) Dynamic source-surface erosion LSIMS uses Cs+ ions sample disolved in non volatile matrix, such as glycerol 15

16 31 FAB: fast atom bombardment (organic applications) the surface is bombarded chiefly by neutral atoms/molecules of argon or preferably xenon Liquid matrix as employed in FAB and LSIMS= non volatile It 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 32 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 different ingredients, including the drug itself, within the tablet. 16

17 Cl 14 2 H 15 Cl 3 H 4 5 SiMe 3 1 EI f CI+ y MALDI- CI-? MALDI+ Comments EI will work but probably not show M+ ([M-57] = most likely - why?); pos CI: hope for NH4+ addition at ; CI neg: hope for electron capture at halogen; ESI+ would need NH4+ (or similar) added to assist ionisation. 2 f y y 3 y :)? CI with ammonia good; EI probably K if molecule doesn t fragment; ESI may struggle with such low MW. 4 :)? CI with methane K, ammonia would not work 5 f y CI with methane K, ammonia would not work 13 :)? EI best; e- capture may be possible in negative mode under appropriate conditions 14 y? Ammonia CI bad with halogens; whatever mode is used to avoid use of methanol! (will react with Cl here 15 y y y ESI+ with NH4Ac (or similar) added Cl 14 2 H 15 Cl 3 H 4 5 SiMe 3 1 EI CI+ MALDI- CI- MALDI+ Comments EI will work but probably not show M+ ([M-57] = most likely - why?); pos CI: hope for NH4+ addition at ; CI neg: hope for electron capture at halogen; ESI+ would need NH4+ (or similar) added to assist ionisation. 2 3 CI with ammonia good; EI probably K if molecule doesn t fragment; ESI may struggle with such low MW. 4 CI with methane K, ammonia would not work 5 CI with methane K, ammonia would not work 13 EI best; e- capture may be possible in negative mode under appropriate conditions 14 Ammonia CI bad with halogens; whatever mode is used to avoid use of methanol! (will react with Cl here 15 ESI+ with NH4Ac (or similar) added 17

18 35 Ionization Methods Gas-phase Solid-state: Liquid-phase Electron Impact (EI) Chemical Ionization (CI) Field ionization (FI) Field desorption MALDI (matrix-assisted laser desorption/ionization) SIMS (Secondary ion mass spectrometry) Plasma desorption Fast atom bombardement (involatile liquid matrix) Electrospray (ESI) Atmosferic pressure chemical inization (APCI) Atmosferic pressure photoionization (APPI ) ASAP Direct analysis in real time ( DART ) Desorption electrospray ionization (DESI) 36 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 Group 2 Group 3 Group 4 ESI APPI APCI ASAP -what type of ion is being formed? -Why is this technique so important? Which type of molecules can be analyzed? 18

19 37 Ionization methods- Liquid phase : Atmospheric pressure ionization Soft ionization Mass spectra provide mainly molecular weight information. Highly efficient production of ions. Different types : ESI, APCI and APPI 38 Ionisation method : Electrospray ESI 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 rganometallic salts N fragmentation, but possible side reactions Polar molecules or molecules with heteroatomes H M MH + H H H H 19

20 39 Ionisation method : Electrospray ESI 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. NH4Ac 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.. 20

21 41 ESI Singly Charged M/W = 281 [M+H] + [M+Na] + [M+NH 4 ] + Courtesy of EPSRC National Mass Spectrometry Service Center 42 ESI Multiple Charging ~16,700 Daltons Myoglobin : oxygen transporting protein Courtesy of EPSRC National Mass Spectrometry Service Center 21

22 43 ESI Multiple Charging Mass spectrum of Myoglobin, observed in the form [M+nH] n ~808 Da 21*808=16968 Da (this carries +21H) = 16947Da m/z Courtesy of EPSRC National Mass Spectrometry Service Center 44 Solvent Effects [M+MeH+Na] + [M+H] + [M+MeH+H] + [M+H] + [2M+H] + Courtesy of EPSRC National Mass Spectrometry Service Center 22

23 45 46 Information from Electrospray MW : 501 No Molecular ion (M+H) + nly ammonium adduct (M+NH4) + MW : MW NH 4 : 18 Ion found : 519 ammonium adduct Courtesy of EPSRC National Mass Spectrometry Service Center 23

24 47 APCI : Atmospheric pressure chemical ionization Advantages Molecular species Insensitive to salts Interface to HPLC Normal phase solvent High flow rates Disadvantages Volatile sample Thermal stability 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. 48 APPI : Atmospheric pressure photoionization Photon source photoionize vapor molecules upon exit from the vaporizer Requires right combination of uv lamp, solvent, analyte and sometime dopant (for example toluene) Positive ions [M+H] +, M* + Rule of thumb, recommended for compounds with a UV active moiety Source-(APPI)/Pages/gp2294.aspx 24

25 49 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) 25

26 51 Natural Isotopes and their relative amount in nature A A + 1 A + 2 Element Element Mass % Mass % Mass % Type H A C A + 1 N A A + 2 F A Si A + 2 P A S A + 2 Cl A + 2 Br A + 2 I A Carbon isotopes - examples C1 C10 C 1 C 10 % % % C1000 C100 C C %

27 53 Ionization Methods Gas-phase Solid-state: Liquid-phase Electron Impact (EI) Chemical Ionization (CI) Field ionization (FI) Field desorption MALDI (matrix-assisted laser desorption/ionization) SIMS (Secondary ion mass spectrometry) Plasma desorption Fast atom bombardement (involatile liquid matrix) Electrospray (ESI) Atmosferic pressure chemical inization (APCI) Atmosferic pressure photoionization (APPI ) Direct analysis in real time ( DART ) Desorption electrospray ionization (DESI) 54 DESI: Desorption electrospray ionization ESI + Direct probe exposure = DESI Principles is : Solvent ionized by ESI is sprayed on the analyte surface Main advantage is able to map compounds position on the native surface Mechanism is not yet established 27

28 55 DART: Direct analysis in real time Direct detection of chemicals on surface, in liquids or in gases without the 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 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 28

29 57 Courtesy of EPSRC National Mass Spectrometry Service Center 58 Ionization Methods: Combined with Chromatography Courtesy of EPSRC National Mass Spectrometry Service Center 29