Co-funded by the Horizon 2020 program of the European Union User school lecture Atmospheric pressure ionization techniques for high resolution mass spectrometry of complex samples Martin Sklorz, Anika Neumann, Christopher Rüger, Ralf Zimmermann Analytical Chemistry University of Rostock 1
Motivation FT-ICR mass spectrometry High accuracy in mass determination Sum formula and isotopic fine structure accessible Universal, as every molecule has a mass, but mass spectrometry detects ions (mass per charge) Ionization requests for complex mixtures: Universal mass spectrometric signals reflects composition of neutrals Preserve molecular ion information No fragmentation or adduct formation (can be done later ) Low matrix effects and linear response 2
Excurse EI: Universal electron impact ionization Universal and simple! In high vacuum, no collision and therefore no chemical reaction, simply: M + e - M + + 2e - 70 ev electron energy Ionization potential for most organic compounds: 7-12 ev Needs transfer of neutrals into high vacuum Excess energy often leads to fragmentation (m/z values <200) often gain of structural but loss of molecular ion mass information 3
ESI APCI APPI APLI Ions were guided into the ICR-cell by gas flow (ion capillary), and static/alternating fields (lenses, funnels, multipols) Neutrals were pumped off (1000 mbar 10-10 mbar) Ion source 1 bar 10-2 mbar 3 mbar 10-2 -10-5 mbar 10-6 mbar 10-7 mbar 10-10 mbar 4
Atmospheric pressure ionization in general Common to all atmospheric pressure ionization techniques High sensitivity obtainable, as high pressure leads to high density of analyte ions Mean free path (N 2 @ 1013mbar, 273K): ~75nm Time between collision: ~200ps Mean residence time in the ion source ~10ms! More than 10 millions of collisions Reactions can/will occur Collisional cooling leads to less fragmentation Processes under collision controlled regimes are not as simple as in high vacuum 5
Electrospray Ionization (ESI) ESI APCI APPI APLI John B. Fenn: Science 1989, Mass Spec Rev 1990, JAMS 1993, Noble prize 2002 6
ESI APCI APPI APLI 7
ESI = solution chemistry ESI APCI APPI APLI HA: Water, methanol, dichloromethane Common modifiers: 0.1-1 % formic acid 0.1-1% acetic acid <0.05% trifluoro acetic acid (toxic!) B: Water, methanol, etc Common modifiers: < 10mM ammonium hydroxide or buffers e.g. ammonium format or ammonium acetate Polar and acid/base - charged molecules are detectable! 8
ESI = solution chemistry Pros: ESI APCI APPI APLI No evaporation necessary even nonvolatile and fragile compounds are ionized Multiple charged ions available high molecular weight compounds accessible works well for polar compounds most suitable for peptides, proteins, and lipids Problems: Molecule-ion-interaction in liquid Adduct formation (Na +, K + ) e.g. from sample or glassware Cluster formation ([M+Na+CH 3 CN] +, [M+H+MeOH] +, [M+H+M] + High matrix suppression all analytes compete to limited (surface-)charge Complex spectra may be obtained (adducts, multiple charge states, etc.) Quantitative results difficult (use internal standards!) Interferences and contaminants compare: Keller et al. Anal Chim Acta 2008 9
ESI APCI APPI APLI ESI+ of Arginine solution cluster formation and sodium adducts Intens. [%] 100 349.232 2xArg-H + ESI_pos_4M_Arginine_000002.d: +iscid MS 80 60 40 20 [%] 0 0 [%] 100 Arg-H + 1914 175.11909 175.11 3xArg-H + Arg-Na + 3xArg-Na + 4xArg-H + 4xArg-Na + 197.10115 2xArg-Na + 371.21434 523.34637 545.32880 200 300 400 500 600 700 ESI 800 ESI_pos_4M_Arginin_acid_180305_000001.d: A i i id900 000001d i+iscid 1000 CIDMS m/z MS 697.46201 0.1 % acid 80 60 40 349.23247 20 523.34712 697.46306 871.58141 0 200 300 400 500 600 700 800 900 1000 m/z 10
ESI APCI APPI APLI ESI+ of Arginine solution declustering ESI_pos_4M_Arginine_000002.d: +iscid MS 349.232 Intens. [%] 100 80 523.34637 371.21434 175.11909 197.10115 697.46201 545.32880 200 300 400 500 600 700 ESI 800 4M A i i id 900 180305 000001d i 1000 CIDMS m/z 35 V CID 175 1914 60 40 20 0 0 [%] 100 80 349.23208 60 371.21429 40 523.34625 197.10113 871.57853 719.44434 697.46176 567.31115 545.32868 393.19652 402.14423 360.22082 219.08321 20 0 200 300 400 500 600 700 800 900 1000 m/z 11
Atmospheric pressure ionization in general Declustering by: - adjusting dry gas flow, - enhancing ion collision energy Ion source 1 bar 10-2 mbar 3 mbar 10-2 -10-5 mbar 10-6 mbar 10-7 mbar 10-10 mbar 12
ESI APCI APPI APLI ESI+ of complex samples effect of concentration ESI_pos_4M_crude_000002.d: +iscid MS 251.14481 x10 7 Intens. dimerisation in complex samples can be troublesome especially for unknown mixtures 298.25421 312.27003 326.28582 340.30160 352.30182 6 270.22261 281.19206 239.14469 217.10516 397.22313 411.23903 425.25492 439.27067 453.28662 467.30257 479.30284 493.31866 507.33468 521.35052 537.38226 551.39834 565.41411 575.39851 591.43011 605.44650 617.44638 4 378.31777 894.19867 763.52773 706.60489 720.62084 657.17055 666.57216 678.57320 636.52436 173.07865 183.08167 197.09742 2 995.88397 959.26931 934.61735 861.63926 875.74733 805.57677 817.57576 ESI_pos_4M_crude_000001.d: +iscid MS diulution 0 x10 8 2.0 393.31272 449.37662 457.35542 473.35080 404.33532 413.27058 421.34461 429.32329 217.10565 225.12949 237.12961 251.14544 267.17699 281.19285 295.20872 309.22452 323.24047 338.24067 351.27230 364.30317 376.30352 1.5 1.0 647.08788 657.17436 545.41012 561.40594 493.32078 501.38261 517.37846 839.19639 765.17278 691.15064 605.43403 617.12862 173.07893 183.08208 197.09793 0.5 0.0 200 300 400 500 600 700 800 900 1000 m/z 13
ESI APCI APPI APLI APCI - evaporated liquid APCI - gas Capillary Corona needle Heated Interface GC-Oven Ion source Heated N 2 (nebulizer gas) Horning et al., Anal Chem 1973,, McEwen et al.jasms 2005, Schiewek et al. ABC 2008 14
ACPI = gas-phase chemistry ESI APCI APPI APLI H 2 O N 2 Charge Transfer N 2 2 N 2 M O 2 N + 2 M M + N 4 + M + Corona discharge needle N + 2 N + 4 H 2 O H 3 O + H 2 O + + HO H 2 O M [M+H] + Protonation 15
ESI APCI APPI APLI Ionization energy Proton affinity Molecule IE (ev) Nitrogen 15.58 Oxygen 422 Carbon dioxide 13.78 Nitrogen 495 Water 12.62 Carbon dioxide 541 Acetonitrile 12.20 n-hexane 672 Oxygen 12.07 n-heptane 677 Methanol 10.84 n-octane 684 Isopropanol 10.17 n-hexane 10.13 Heptane 9.93 Isooctane 9.80 Benzene 9.24 Furan 8.88 Toluene 8.83 Anisole 8.20 Naphthalene 8.14 Triethylamine 7.53 Molecule webbook.nist.gov, Hunter&Lias J Phys Chem Rev Data 1998, Hunter&East J Phys Chem 2002 Water 691 Benzene 750 Methanol 754 Toluene 784 Isopropanol 793 Acetonitrile 794 Naphthalene 803 Furan 804 Anisole 840 Triethylamine 982 PA kj/mol 16
APCI = gas-phase chemistry Pros: ESI APCI APPI APLI No formation of Na and K adducts Higher linear dynamic range and less matrix effects (compared to ESI) Ideal for hyphenation to gas and liquid chromatography works well for intermediate polar compounds most suitable for small molecules (metabolomic, petroleomic) Problems: Lossless evaporation needed not applicable for thermo-labile components Molecule-ion-interaction in gas-phase Complex gas-phase chemistry (reactants, solvent, concentration, reaction time ) Corona current needs to be tuned, position of sprayer and needle is critical Adduct formation with oxygen 17
ESI APCI APPI APLI Example: Fingerprinting asphaltene pyrolysis products 359.276784 413.323746 511.433264 APCI: CHS-, CHS 2 -, CHSO x - and CH-species dominating 567.495669 200 300 400 500 600 700 m/z > 3,500 distinct peaks Pyrolysis GC/EI-MS results: Mainly alkanes and alkenes, and only minor concentration of alkylated benzothiophenes 457.165160 CHSO 2 457.201650 CHS 2 457.255968 CHO CHSO CH 457.292451 CHS CHS 2 CHSO 2 CHSO 457.313407 CHO 457.15 457.20 457.25 457.30 457.35 457.40 457.45 m/z 457.349666 457.365837 457.386329 CHN CH CHS CHS 457.440395 CHS 2 457.476707 CHS 2 18
ESI APCI APPI APLI APPI - liquid APPI - gas VUV Lamp Capillary Heated Interface VUV Lamp GC-Oven Ion source Heated N 2 (nebulizer gas) Horning et al., Anal Chem 1973, McEwen et al. JASMS 2005, Benter et al. ABC 2008 19
ESI APCI APPI APLI APPI = direct photon/molecule reaction Direct Photo Ionisation hv M M + + e - if IE(M) < hv Gaseous Phase Reactions S hv S + + e - S + + M S + M + if IE(M) < IE(S) S + + M [S-H] + [M+H] + if PA(M) > PA(S) + gas-phase chemistry (by dopant or solvent or matrix)!!! 20
Ionization energy Molecule IE (ev) ESI APCI APPI APLI Nitrogen 15.58 Carbon dioxide 13.78 Water 12.62 Acetonitrile 12.20 Oxygen 12.07 Methanol 10.84 Krypton lamp (116 124nm) 10eV 10.6eV Isopropanol 10.17 n-hexane 10.13 Heptane 9.93 Isooctane 9.80 Benzene 9.24 Furan 8.88 Toluene 8.83 Anisole 8.20 Naphthalene 8.14 Triethylamine 7.53 webbook.nist.gov, Short et al. JASMS 2006 Direct PI M + hv M + + e - Xenon lamp (148nm) 8.4eV Common solvents (and oxygen) are not ionized, but may strongly absorb photons n-c 6 H 14 CH 3 CN 0 5 10 15 21 absorption cross section (Mb) 200 100 200 200 200 N 2 O 2 CCl 4 248nm 124nm 84nm photon energy (ev)
ESI APCI APPI APLI APPI = direct photon/molecule reaction and gas-phase chemistry Pros: Higher linear dynamic range and less matrix effects (compared to APCI) Low background noise Ideal for hyphenation to gas chromatography works well for compounds with comparable low IP most suitable for small and even non-polar molecules Problems: Lossless evaporation needed not applicable for thermo-labile components Still molecule-ion-interaction occurs (formation of radical cation vs. protonation) Dopants and solvents may lead to complex gas-phase chemistry Adduct formation with oxygen Lamp has a limited lifetime Syage_et al. Am Lab 2000, Kauppila et al. Anal Chem 2002, Hanold et al. Anal Chem 2004, Kauppila, Syage, Benter Mass Spec Rev. 2017 22
ESI APCI APPI APLI Example:Fingerprinting asphaltene pyrolysis products Rüger et al ASMS2018 23
ESI APCI APPI APLI APLI - evaporated liquid APLI - gas Laser beam Capillary Heated Interface Laser beam GC-Oven Ion source Heated N 2 (nebulizer gas) Schmidt et al. Anal Chem 1999, Kersten et al. JASMS 2011 24
ESI APCI APPI APLI APLI (REMPI) uses UV photons 248nm (5.00 ev) 266nm (4.66 ev) M*+hv M + High photon density (~10 7 W/cm 2 ) Short pulses (6ns 10ns) Two photon resonant process Selective ionization of aromatic and polyaromatic compounds M+hv M* 1. UV photon adsorbed 2. Lifetime of intermediate longer than pulse length (2. photon) 3. 2 x hv > IE Boesl et al. Int J Mass Spec Ion Process 1994, Zimmermann et al RCM 1996 25
ESI APCI APPI APLI APLI = direct photon/molecule reaction Pros: Higher linear dynamic range and less matrix effects (compared to APPI) Compatible to common LC-solvents High selectivity and extreme high sensitivity can be reached works well for compounds with aromatic core (low IE) most suitable for polycyclic aromatic hydrocarbons and derivatives Problems: Lossless evaporation needed not applicable for thermo-labile components Ionization efficiency depends strongly on compound structure, laser wavelenght, and laser fluence Laser safety Constapel et al. RCM 2005, Schiewek et al Anal Chem 2007, Brinkhaus et al. ABC 2017 26
ESI APCI APPI APLI Intens. x10 7 1.0 APCI C 20 H 12 +H + 253.1018 GC_APCI_M3_mix_1_100_180629_000001.d: +MS, Profile, 36.7min #2213, Background Subtracted Benzo[a]pyrene C 20 H 12, m/z=252.0939 0.8 0.6 0.4 0.2 0.0 x10 7 5 4 3 2 1 0 248.0625 C 20 H 10 + 250.0784 APLI (266nm) C 20 H 10 + C 20 H 12 + 250.0783 251.0816 252.0941 254.1052 1.00 + 0.75 C 20 H 12 252.0940 13 C 1 12 C 19 H 12 + 253.0973 13 C 1 12 C 19 H 12 +H + 13 C 2 12 C 18 H 12 + Intens. x10 7 GC_APCI_M3_mix_1_100_180629_000001.d: +MS, Profile, 36.7min #2213, Back 253.1018 GC_APLI_M3_mix_1_100_180628_000001.d: +MS, Profile, 36.65min #2220, Background Subtracted 0.50 0.25 0.00 x10 7 1.25 1.00 0.75 0.50 0.25 13 C 1 12 C 19 H 12 + GC_APLI_M3_mix_1_100_180628_000001.d: +MS, Profile, 36.65min #2220, Bac 13 C 1 12 C 19 H 12 + 253.0973 C 20 H 12 +H + 247.5 250.0 252.5 255.0 257.5 260.0 262.5 265.0 267.5 270.0 m/z 0.00 253.08 253.09 253.10 253.11 253.12 m/z 27
Summary ESI* APCI APPI APLI Direct formation of radical cations by charge transfer or photoionization Proton transfer and chemistry in gas or liquid phase Additional chemistry by dopant assisted Ionization Reactions can/will take place and depend on: reactant concentration reaction enthalpies and rates fluid dynamics (flows and source design) 28
Summary Ionization method determines the detectable components molecular weight (vaporizable?) polarity (internal acidic/basic ions?) and reactivity Ionization potential and proton affinity 29
Ionization requests for complex mixtures Universal mass spectrometric signals reflects composition of neutrals NO Ionization efficiency varies extremely by: The applied technique Physical and chemical properties of the targeted compounds Solvent, background and matrix components and modifiers 31
Preserve molecular ion information No fragmentation or adduct formation Low matrix effects and linear response YES (mostly ) Diluting the sample Using pure solvents and gases Optimizing solvent composition and instrumental settings Clean-up and separation steps before mass spectrometric analysis Analyzing the same sample with different dilution ratios Spiking the sample or adding isotope labeled standards Checking the ratio of radical cation / protonated species. Think about: Ionization requests for complex mixtures Rem: Additional fragmentation and adduct formation may happen during ion transport from atmospheric pressure to high vacuum 32
Co-funded by the Horizon 2020 program of the European Union General reading Jürgen H. Gross: Mass spectrometry - A Textbook, Springer International Publishing (2017) John B. Fenn: Electrospray Wings for Molecular Elephants (Nobel Lecture). Angew. Chem. Int. Ed. 42, 3871-3894 (2003): Ralf Schiewek, Matthias Lorenz, Ronald Giese, Klaus Brockmann, Thorsten Benter, Siegmar Gäb, Oliver J. Schmitz: Development of a multipurpose ion source for LC-MS and GC-API MS. Anal. Bioanal. Chem. 392, 87 96 (2008) Tiina J. Kauppila, Jack A. Syage, Thorsten Benter: Recent Developments in atmospheric pressure photoionization-mass spectrometry. Mass Spec. Rev. 36, 423-449, 2017 33